Emerging treatment options for patients with p53-pathway-deficient CLL.

Introduction

Chronic lymphocytic leukemia (CLL) is the most common leukemia in developed countries. 1 Incidence in men exists at nearly a 2:1 ratio compared with women, and the median age at diagnosis is 70 years. 2 The clinical course of CLL is highly variable, as some patients live for decades without complications, while others succumb to the disease within months of diagnosis.

A diagnosis of CLL is made by the presence of ⩾5 × 10 9 /L clonal B cells in the peripheral blood for a minimum of 3 months. 3 Commonly, lymphocytosis is the only manifestation of CLL at diagnosis; patients are frequently otherwise asymptomatic. The consensus standard of care for patients in early stages of the disease is to offer close follow up without initiating anti-leukemia therapy. 3 However, continued proliferation of CD5+ mature-appearing neoplastic cells can result in further leukocytosis, lymphadenopathy, splenomegaly, and autoimmune cytopenias. 4 Patients progressing to these symptomatic stages are then eligible for various treatments.

Over the past 2 decades, our understanding of the pathogenesis of CLL has increased dramatically, leading to the development of new therapeutic strategies for such patients requiring treatment. Treatments have evolved from simple alkylating agents to immune-checkpoint inhibitors, genetically engineered cellular therapies, and targeted molecular agents. From 2010 to 2019, eight new agents have been approved by the US Food and Drug Administration (FDA), marking the most prolific period of drug development for CLL on record. All age groups and risk subtypes of CLL patients have benefited from this therapeutic renaissance. However, patients with CLL harboring TP53 abnormalities, usually in the form of del(17p) or TP53 mutations, continue to be a challenging population for durable disease control.

TP53 evaluation

Cytogenetics

Cytogenetic evaluation is a routine and relatively straightforward means to risk-stratify patients with CLL, in which few recurrent cytogenetic abnormalities are seen. Approximately 80% of cases will harbor at least one chromosomal aberration at diagnosis. 5 Deletion of the long arm of chromosome 13 [del(13q)] is the most common alteration (~55% of cases), and is associated with favorable prognosis. 5 This is followed in frequency by the intermediate-risk category of cytogenetic abnormalities, which encompasses trisomy 12 (10–20% of cases) and deletions in the long arm of chromosome 11 [del(11q); ~15% of cases]. Deletions in the short arm of chromosome 17 [del(17p)] are found in 5–8% of cases at diagnosis, and have been associated with high-risk disease. 5 Indeed, numerous prospective clinical trials have confirmed a poor prognosis for patients with del(17p). 6 – 8 The incidence of del(17p) is even higher in relapsed or refractory CLL (~30% of cases) and confers a similarly dismal prognosis. 9 However, notable therapeutic advances discussed in this review have mitigated the poor outcomes associated with del(17p).

A critical gene within the deleted portion of chromosome 17p is TP53 , which encodes for the tumor suppressor p53. p53 is often referred to as the ‘guardian of the genome’ in reference to its critical role in maintaining genomic integrity. 10 Abrogation of wild-type p53 functions allows cells to accumulate mutations, due to loss of p53-dependent apoptotic and cell-cycle arrest functions, and often results in malignancy. Indeed, germline TP53 mutations are a hallmark of Li-Fraumeni syndrome, wherein patients develop malignancies of various anatomic sites (including hematologic), often before age 30. 11

TP53 mutations

Somatic mutations in TP53 are observed in CLL in ~10% of cases at diagnosis and are often associated with del(17p). Indeed, almost 80% of cases with del(17p) will harbor a TP53 mutation in the remaining allele, a phenomenon referred to as loss of heterozygosity (LOH). 12 Importantly, mutations in TP53 have also been observed in the absence of 17p deletions and have been associated with poor prognosis, even in this setting. 13 , 14 Such prognoses are evolving in the current era of targeted therapeutics as discussed in this review.

Function

At its simplest, p53 is a transcription factor. In normal cells under basal conditions, the p53 protein exists at levels often below the threshold of antibody-based detection methods. This is largely due to the presence of negative regulators, including mouse double-minute 2 homolog (MDM2). MDM2 binds p53 and inhibits its transcriptional activity by interfering with its interaction with the transcriptional machinery. 15 However, most importantly, MDM2 also ubiquitinates p53, thereby targeting p53 for proteasomal degradation. 16 – 18 In response to cellular stresses, such as deoxyribonucleic acid (DNA) damage or deregulated oncogene activation, this MDM2-mediated negative regulation of p53 is suppressed, allowing for p53 stabilization and transcription of p53 target genes that drive apoptotic and cell-cycle arrest programs.

Classic chemotherapeutic agents exert cytotoxic effects by directly or indirectly inducing DNA damage. Such genomic injury activates a group of protein kinases that include ataxia–telangiectasia mutated (ATM; in the case of double-strand DNA breaks) and ATM and Rad3 related (ATR; in the case of single-strand DNA breaks or replication stress). 19 These kinases begin a cascade of events that include the phosphorylation of both MDM2 and p53. The result is decreased interactions between MDM2 and p53, thus preventing ubiquitination of p53 and thereby facilitating p53 stabilization. 20 – 22 As p53 accumulates in the stressed cell and is further phosphorylated, it exerts transcriptional activity, inducing expression of genes that either promote cell-cycle arrest, senescence, or apoptosis. For example, p53-mediated induction of the CDKN1A gene, which encodes the p21 protein, arrests the cell cycle and prevents proliferation of cells that have damaged DNA that could promote tumor formation or further tumor evolution. 23

Among p53-responsive genes that induce apoptosis are pro-apoptotic members of the B-call lymphoma 2 (BCL2) family, such as BAX, PUMA , and NOXA. 24 – 26 p53 has also been shown to directly decrease levels of the antiapoptotic protein BCL2. 27 , 28 Together, the BCL2 family controls the release of cytochrome c from the outer membrane of mitochondria, resulting in an irreversible apoptotic cascade. 29 Thus, upon administration of many chemotherapeutic agents, the desirable result of cell death results in eradication of some or all of the tumor.

In scenarios where p53 cannot bind DNA or cannot be stabilized in order to act as a transcription factor, the desirable cytotoxic effects of chemotherapy are not evident. Importantly, the damage induced by the chemotherapy may also go unrepaired in the absence of wild-type p53 function. Thus, administration of such drugs may accelerate the accumulation of genotoxic insults while failing to induce cell death. This sets the stage for a challenging therapeutic scenario.

Consequences of TP53 aberrations

In CLL (as well as many other malignancies), these normal wild-type functions of p53 are interrupted. As previously described, in addition to 17p deletions, TP53 can also be mutated in CLL. These are often missense mutations that predominantly occur in the DNA-binding domain of TP53 and result in the loss of wild-type p53 functions. 30 Indeed, mice that lack Trp53 (the murine homolog of TP53 ) or express a mutant form (e.g. R172H, R172P, or R270H, corresponding to the R175 and R273 ‘hotspot mutations’ in humans) are tumor prone, often developing tumors of T- or B-cell lineage. 31 – 34 Interestingly, some of these mutations have been shown to gain oncogenic activity as they alter the cellular transcriptome. This has been reviewed elsewhere. 35 This review will remain focused on the deficiencies of wild-type p53, which confer a fitness advantage to malignant cells.

Even when TP53 or chromosome 17p are not directly influenced, alterations in p53 regulators may likewise render the p53 pathway deficient. Overexpression of MDM2 has been observed in B-cell malignancies, including CLL. 36 , 37 A single-nucleotide polymorphism in MDM2 has also been linked to poor outcomes in CLL. 38 , 39 Likewise, overexpression of MDM4 , another negative p53 regulator, has been observed in a subset of p53-wild-type CLL that responds poorly to MDM2 inhibition. 40 These observations have been validated in murine models, where increased levels of the p53’s negative regulators MDM2 or MDM4 result in hematologic tumors. 41 , 42

Conversely, decreased expression of MDM2 inhibitors can also abrogate p53 functions. For example, p14 ARF , which is encoded from an alternative reading frame in CDKN2A , binds to MDM2 and inhibits MDM2-dependent ubiquitination of p53. 43 – 45 As such, mutations, deletions, and hypermethylation of the CDKN2A locus have been associated with aggressive disease courses in CLL. 46

Together, these data underscore the importance of p53 deregulation in hematopoietic oncogenesis and response to conventional chemotherapies. These observations also provide substantial rationale for utilizing therapeutic modalities that function without a requirement for wild-type p53.

Treating CLL

Initiation of therapy for CLL is not required for all patients at the time of diagnosis. While cytogenetic [del(17p)] or molecular ( TP53 mutation) aberrations may confer a worse prognosis, the presence of these events alone is not an indication for treatment initiation. Patients may start treatment based on disease-related symptoms or evidence of disease progression, such as physical findings of bulky lymph nodes or splenomegaly, or progressive lymphocytosis or cytopenias. Guidelines for initiation of therapy have been reviewed by the International Workshop on CLL. 3 Outside of clinical trials, early therapy is generally not recommended before these criteria are met, as there is currently no evidence that starting chemotherapy earlier in asymptomatic patients improves survival in CLL. 47 However, early use of targeted molecular agents is an active field of study. 48 , 49

Historic treatments-genotoxic chemotherapies

Alkylating agents

Chlorambucil is an alkylating agent that was approved for medical use in 1957. It exerts its anticancer effects via interference with DNA replication, leading to cellular apoptosis in a p53-dependent manner. 50 The original studies using chlorambucil plus prednisone for treatment of CLL had modest overall response rates (ORRs) and complete response (CR) rates of 47% and 7%, respectively. 51 Subsequently, patients would almost invariably progress with splenomegaly and bulky lymphadenopathy, the majority of whom succumbed to their disease or due to treatment complications.

Chlorambucil is still widely used in Europe and in elderly patients for palliative disease control. 52 In a meta-analysis of seven clinical trials of the German CLL study group, of 3552 patients, 77% received chlorambucil plus anti-CD20 antibody therapy. 53 Notably, 152 patients were more than 80-years old at treatment initiation. 53 Unfortunately, chlorambucil was especially poor in controlling CLL with TP53 abnormalities, as these patients had significantly inferior ORR (27% versus 87%), shorter progression-free survival (PFS; 5-year PFS: 5% versus 17%), and overall survival (OS; 5-year OS 20% versus 59%) compared with patients without TP53 abnormalities. 12 , 54 Therefore, chlorambucil is currently not recommended for cases of CLL with TP53 abnormalities, except for palliative purposes. These observations align with our biologic understanding of the consequences of administering such therapies to malignancies deficient in wild-type p53.

Purine analogs

Fludarabine is a purine analog discovered in the early 1970s as having cytotoxic activity in vitro . 55 In a seminal paper by Rai and colleagues, fludarabine showed increased ORR (63% versus 37%), CR (20% versus 4%), and PFS (25 months versus 14 months) compared with chlorambucil. 56 However, further analysis of patients with TP53 abnormalities demonstrated continued poor outcomes compared with standard-risk patients. 56 Approximately 9% of CLL patients had TP53 abnormalities at diagnosis, but by the time of relapse, 44% of patients had TP53 abnormalities, suggesting that fludarabine exerts a selection advantage for TP53 aberrant clones. 12 , 57 Nonetheless, fludarabine has been a backbone of frontline CLL chemotherapy since Rai and colleagues’ publication in 2000, and its combination with the cytotoxic agent cyclophosphamide, and the monoclonal antibody rituximab have been the genesis of immunochemotherapy (e.g. fludarabine–cyclophosphamide–rituximab, FCR) in hematologic malignancies.

Bendamustine

Bendamustine is a multifunctional alkylating agent containing a purine-like ring that has demonstrated clinical activity in multiple hematologic malignancies. In addition to causing DNA damage responses, this drug has been observed to trigger intrinsic apoptotic pathways, including upregulation of p53 upregulated modulator of apoptosis (PUMA) and phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) and increasing expression of mitochondrial apoptogenic proteins. 58 In addition, bendamustine can cause necrotic cell death, and thus shows some efficacy in cells lacking functional p53 or apoptotic pathways. 59

Bendamustine has been studied in the frontline setting for patients with newly diagnosed CLL. In one study, del(17p) was noted in 7% of patients and the median PFS was less than 12 months, consistent with previous reports using genotoxic chemotherapy approaches. 60 Bendamustine showed a promising response rate and was better tolerated than FCR, which led to community practices, particularly in the US, to adapt to using bendamustine, particularly for elderly patients who were not eligible for clinical trials. These practices led to a head-to-head study in which it was determined that, in combination with rituximab, bendamustine has a role in the frontline setting for patients lacking del(17p) who are not fit to receive FCR. 61

Antibody therapy

Rituximab

Proteins expressed on the surface of leukemia cells represent a bevy of biologic targets. CD20 is a cell-surface protein expressed on pre-B cells and on differentiated B cells of both normal and malignant origin. 62 The anti-CD20 antibody rituximab was the first of the targeted antibody therapies to change treatment paradigms for all B-cell malignancies. A combination of signaling-induced cell death, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity is responsible for rituximab-induced cell death. 63 These distinct mechanisms of action and manageable side-effect profile allowed for straightforward combinations with already existing chemotherapeutic agents. 64 FCR was effective in relapsed CLL and set the standard for frontline therapy for newly diagnosed, fit CLL patients requiring treatment. 65 , 66 With this combination, ORR improved to 92% with a CR of 70%. 66 However, even though this treatment modality has proven effective in many patients with CLL, those with TP53 abnormalities continued to have poorer response rates and shorter remission durations.

Ofatumumab

Ofatumumab is also an anti-CD20 antibody that targets a distinct epitope on CD20 that is not targeted by rituximab. 67 In fludarabine-refractory CLL patients, ofatumumab was safe and effective as a single agent, with ORR of 58%, PFS of 5.9 months, and OS of 15.4 months. 68 While adverse events were minimal, routine use of ofatumumab has not been adapted since it has not demonstrated superiority over rituximab.

Obinutuzumab

Obinutuzumab is another CD20 antibody that was first studied in combination with chlorambucil for elderly patients who were not candidates for standard chemoimmunotherapy. 69 Patients receiving both drugs had significantly higher CR rates compared with chlorambucil alone (20.7% versus 7%). 69 However, patients with del(17p) did not see such benefit. Importantly, the addition of obinutuzumab did not elicit substantial toxicity, a crucial consideration in these patient populations. 69 The results of this study led to trials combining obinutuzumab with fludarabine–cyclophosphamide (FC) or bendamustine, where the combinations demonstrated robust activity, though only one patient harbored del(17p). 70 For obinutuzumab–FC, the ORR was 62% with 24% CR/CRi with incomplete hematologic recovery (CRi). Obinutuzumab–bendamustine had an ORR of 90% with 45% CR/CRi. 70 Further studies are ongoing to evaluate which patient populations are best to incorporate this novel CD20 antibody and to investigate its role in p53-pathway-deficient CLL.

Alemtuzumab

Alemtuzumab is an anti-CD52 antibody that was designed for CLL patients refractory to both alkylating and purine analog agents; a population usually harboring TP53 abnormalities. 71 In the early 2000s, fludarabine-refractory patients had an anticipated survival of only 10 months. This led to the design of a trial treating high-risk CLL patient populations (fludarabine-refractory or harboring TP53 abnormalities) with alemtuzumab. This trial demonstrated an ORR of 33%, but came with significant infectious complications, including cytomegalovirus reactivation and fungal infections. 72 , 73 Alemtuzumab is still available for CLL with abnormal TP53 ; however, its use has been limited due to this significant toxicity.

Targeted agents

To better treat CLL with deficiencies in the p53 pathway, using agents that kill cells independently of p53 is imperative. Understanding the mechanism of action of currently available targeted agents provides rationale for their utilization and combinations.

B-cell expansion in CLL is largely driven by constitutive B-cell receptor (BCR) signaling. 74 The BCR signals through a series of tyrosine kinases, including phosphatidylinositol 3-kinase delta (PI3Kδ) and Bruton’s tyrosine kinase (BTK). 75 Understanding the BCR signaling axis has been pivotal in developing effective drugs for CLL. To this end, PI3Kδ inhibitors (idelalisib, duvelisib, and umbralisib) and BTK inhibitors (ibrutinib, acalabrutinib, and zanubrutinib) are in various stages of clinical use. These drugs directly inhibit the pathway that produces and maintains B cells of both normal and malignant origin.

In addition to constitutive BCR signaling, CLL cells often harbor high expression of BCL2, an antiapoptotic protein that sustains cell survival. 76 , 77 As such, methods that inhibit or decrease levels of BCL2 in CLL cells have been shown to activate apoptosis. 78 This understanding led to development of venetoclax, a BCL2 inhibitor, in CLL.

Together, these drugs have been revolutionary, as they inhibit critical mediators of CLL emergence and survival. They are discussed individually in the following section. The major clinical trials utilizing these drugs in patients with p53-pathway-deficient CLL are summarized in Table 1 .

Idelalisib

Idelalisib is an orally available small molecule that specifically inhibits the catalytic subunit of PI3Kδ. 79 Mice lacking this isoform have severe B-cell defects, highlighting the importance of this specific protein in the development and function of B cells. 80 – 82 In CLL, PI3Kδ signals downstream of the BCR, leading to constitutive activation of the PI3K pathway and upregulation of antiapoptotic proteins. 83 , 84 Chronic BCR signaling subsequently leads to chronic activation of PI3Kδ in B-cell malignancies, including CLL. 85 Downstream of PI3Kδ lies a number of effector molecules, including AKT (protein kinase B). This is particularly relevant in the context of p53, as AKT phosphorylates MDM2, enhancing its ability to ubiquitinate p53 and target it for proteasomal degradation. 86 Thus, in addition to blocking the upregulation of antiapoptotic proteins, inhibition of PI3Kδ may partly restore p53 activity by allowing p53 stabilization. Indeed, in other cancer models, idelalisib has been shown to induce PUMA. 87 In the absence of wild-type p53 function, upregulation of PUMA can push cells toward apoptosis. Thus, idelalisib can be useful in treating malignancies with chronic BCR activation, even in the setting of p53 pathway deficiency.

Idelalisib is currently FDA approved for relapsed CLL when used in combination with rituximab. This approval was based on the double-blind, placebo-controlled phase III study using idelalisib plus rituximab versus placebo plus rituximab. The median PFS in the idelalisib plus rituximab group was 20.3 months, whereas the placebo plus rituximab group had a median PFS of 5.5 months. 88 , 89 OS showed similar trends of 40.6 months versus 34.6 months. 89 This study enrolled a challenging patient population, as more than 40% of patients had del(17p), and the majority had received FCR and had a median of three prior therapies. In long-term follow up, where all patients received idelalisib after initial treatment, patients with del(17p) or TP53 mutations had an OS of 28.5 months in the idelalisib–rituximab arm compared with only 14.8 months in the placebo–rituximab arm. 89

While clinically effective, many patients do not tolerate idelalisib, and this agent has a more adverse safety profile than other targeted agents, with relatively high rates of diarrhea and infectious pneumonia. 90 The FDA issued a black-box warning after frequent immune-mediated occurrences of colitis, pneumonitis, or hepatotoxicity were reported. 91 Cessation of the drug, in addition to use of corticosteroids, appear to alleviate the immune reaction. 92 Combinations with idelalisib have also been associated with higher rates of infection, including Pneumocystic jirovecii pneumonia. 88 , 93

Duvelisib

Duvelisib is an orally available PI3Kδ inhibitor that demonstrated superiority to ofatumumab in a phase III clinical trial of relapsed refractory CLL patients. 94 In this study, 160 patients were randomized to receive single-agent duvelisib or single-agent ofatumumab. Approximately 30 patients in each group had p53 deficiencies. Duvelisib was able to obtain an overall response rate of 74% versus 45% with ofatumumab alone and a median PFS of 13.3 months versus 9.9 months. The efficacy of duvelisib was maintained in p53-deficient patients, as their median PFS was 12.7 months compared with 9 months for patients receiving ofatumumab. Based on these data, the FDA approved the use of duvelisib in the treatment of relapsed refractory CLL. 94

Umbralisib

Umbralisib is a newer inhibitor of PI3Kδ that is structurally distinct from idelalisib and duvelisib. 95 In a phase I study, 17/20 patients with relapsed refractory CLL had an objective response. 95 Of eight patients with high-risk cytogenetic features, six had a response and none had disease progression. Importantly, umbralisib appeared to have a more favorable safety profile than idelalisib and duvelisib, with fewer incidences of autoimmune-like toxicities. 95 A study evaluating the use of umbralisib in patients who were intolerant to idelalisib or BTK inhibitors suggests that these patients may benefit from switching to umbralisib. 96 Median PFS in this cohort, which comprised 24% p53-deficient patients, was 23.5 months. Importantly, patients with prior PI3Kδ-targeted therapy did not require umbralisib discontinuation, due to recurrence of adverse events that led to discontinuation of the original drug. 96

Ibrutinib

Ibrutinib is a tyrosine kinase inhibitor that specifically inhibits Bruton’s tyrosine kinase (BTK), a critical molecule mediating BCR signaling. In a phase I/II study of ibrutinib in relapsed refractory patients with high-risk disease, ORR was 71%, and an additional 20% of patients had a partial response (PR) with lymphocytosis. The latter was subsequently described as an ibrutinib-related phenomenon that does not have negative clinical impact. At 26 months of median follow up, PFS was 75% and OS was 83%. 97

Ibrutinib was the first novel agent to obtain full FDA approval for frontline treatment (March 2016) and relapsed refractory settings in patients with del(17p) CLL. In the frontline setting, ibrutinib was compared with its combination with rituximab versus bendamustine–rituximab in older patients. 98 Critically, for patients in this study with TP53 abnormalities, the median PFS for the bendamustine plus rituximab group was 6 months compared with the ibrutinib plus rituximab group, where median PFS was not reached at 3 years of follow up. 98

These encouraging results with ibrutinib in the p53-deficient patients were further validated in the RESONATE-17 trial. Ibrutinib in del(17p) populations showed 2-year PFS of 63% and OS of 75% in this trial, which enrolled 144 patients, all with centrally confirmed del(17p). 99

Ibrutinib was also evaluated in the frontline setting in combination with obinutuzumab in a phase III trial. 100 Ibrutinib plus obinutuzumab demonstrated superiority compared with chlorambucil–obinutuzumab, as median PFS was not reached in the ibrutinib group and was 19 months in the chlorambucil–obinutuzumab group. In p53-deficient patients, median PFS was 11.3 months in the chlorambucil–obinutuzumab group but was not reached in the ibrutinib–obinutuzumab group. 100

In the longest follow-up study of single-agent ibrutinib, 5-year follow up still showed an ORR of 89% and CR in 29% in treatment-naïve patients, and CR of 10% in the relapsed refractory population. However, the del(17p) population had a median PFS of 26 months compared with 51 months for the whole relapsed refractory population. 101

Ibrutinib has also been shown effective in an in vivo model of de novo p53-mutated CLL. 102 Importantly, this study also demonstrated that frontline ibrutinib treatment in this model did not place undue pressure on the remaining wild-type Trp53 alleles to undergo LOH. While these findings must be fully substantiated clinically, they are of great interest, since most single trials do not have substantial numbers of patients harboring only heterozygous p53 mutations.

Ibrutinib has been a clear breakthrough in treating CLL patients with TP53 abnormalities and has been incorporated into all guidelines for the frontline and relapse settings. While outcomes with this drug are substantially better than those seen with chemoimmunotherapy in similar populations, ibrutinib alone may not be optimal for overcoming TP53 abnormalities.

Acalabrutinib

Acalabrutinib is a second-generation, more selective, irreversible inhibitor of BTK. This was tested in a phase I/II study of relapsed refractory CLL patients with a median of three prior lines of therapy in which 31% had TP53 abnormalities. The acalabrutinib arm had an ORR of 95%, partial response rate of 85%, and partial response with lymphocytosis in 10%. Interestingly, del(17p) patients had a 100% ORR, and no observations of Richter’s transformation were noted in that trial. 103

In a phase III study, single-agent acalabrutinib was compared with rituximab–idelalisib (IR) or rituximab–bendamustine (BR) in a relapsed refractory population. 104 PFS was not reached in the acalabrutinib arm versus 16.5 months in the IR or BR groups. This improvement in PFS was maintained in the p53-deficient subgroups. 104

Acalabrutinib has also demonstrated efficacy in a small study of patients deemed intolerant to ibrutinib. 105 Patients with del(17p) had an ORR of 67%, and median PFS was not reached in this subgroup at 13.6 months of follow up. 105 Of 33 patients in this study who were previously deemed intolerant to ibrutinib, only 3 discontinued acalabrutinib due to adverse events. 105 Another phase II study has generated similar results, demonstrating that acalabrutinib is tolerable and effective in ibrutinib-intolerant patients. 106 Indeed, a head-to-head comparison of ibrutinib versus acalabrutinib is currently underway for relapsed refractory patients with high-risk CLL [ ClinicalTrials.gov identifier: NCT02477696 ].

Zanubrutinib

Zanubrutinib is an orally available kinase inhibitor with greater selectivity for BTK than ibrutinib. 107 This selectivity is desirable, since the off-target effects of ibrutinib are thought to largely mediate the adverse events observed clinically. 107 In a phase I study, ORR was 100% in patients with p53 abnormalities. 107 This high rate of activity is likely due to the fact that zanubrutinib has a longer half-life compared with acalabrutinib or ibrutinib, thus prolonging exposure of CLL cells to specific BTK inhibition. 107 Zanubrutinib is now being compared with ibrutinib in an ongoing phase III trial of relapsed refractory CLL [ ClinicalTrials.gov identifier: NCT03734016 ]. 108

The evidence for the use of BTK inhibitors in TP53 -aberrant CLL is thus promising, and highlights how targeted agents not requiring the p53 pathway for their efficacy are critical for patients with TP53 alterations.

Venetoclax

Venetoclax is an orally available inhibitor of BCL2, an antiapoptotic protein that sustains cell survival and is highly expressed in CLL. 76 BCL2 binds to pro-apoptotic proteins BIM, BAD, BID, and NOXA, thereby inhibiting initiation of the apoptotic cascade. 109 Inhibition of BCL2 by venetoclax thus allows pro-apoptotic proteins to promote cell death. Intact p53 regulates the expression of several pro-apoptotic proteins such as PUMA and BAX, which promote apoptosis. In the absence of wild-type p53 function, venetoclax still holds promise, as it directly antagonizes BCL2 and promotes cell death in a p53-independent manner. 110

This lack of p53-dependency is highlighted by studies showing that in relapsed refractory CLL patients with TP53 mutations or chromosome 17p deletions, single-agent venetoclax led to a 20% CR rate, with 5% of patients having no minimal residual disease (MRD) via flow cytometry. 111 In this difficult-to-treat population, single-agent venetoclax led to 69% PFS at 16 months. 111

Venetoclax has been studied in combination with obinutuzumab (VO) versus chlorambucil–obinutuzumab (CO) in previously untreated patients. 112 The use of venetoclax was associated with a longer PFS, including in the p53-aberrant group, where 24 month PFS estimates were 73.9% in the VO arm versus 32.7 in the CO arm. 112 Further, 70.8% of patients with aberrant p53 treated with VO achieved MRD negativity in peripheral blood compared with only 9.1% in CO. 112

In relapsed refractory CLL, venetoclax plus rituximab (VR) was compared with bendamustine plus rituximab (BR). 113 In this study, venetoclax was stopped after 2 years of treatment. The 2-year rate of PFS in del(17p) patients was 81.5% in the VR group versus 27.8% in the BR group. 113 Post-treatment follow up of this study confirmed these results, showing that median PFS in the VR group for patients with TP53 mutations was 36 months, versus 12.9 months in the BR group. 114 Likewise, del(17p) patients did not reach median PFS, versus 15.4 months in the BR group. 114 Notably, aberrant p53 was associated with an increased risk of disease progression after venetoclax cessation. 114

Ibrutinib plus venetoclax

Since chronic BCR signaling is exacerbated by overexpression of BCL2, dual inhibition of these molecules is biologically justified. In the frontline setting, the combination of ibrutinib and venetoclax has been studied to combine mechanistically distinct drugs to minimize the risk of disease resistance developing. In patients with high-risk CLL with TP53 aberrations, 80% achieved CR with 61% having no MRD. 115 While longer follow up is needed to determine the durability of the disease response, this treatment combination represents a completely chemotherapy-free regimen that is highly effective in treating patients with TP53 alterations.

Currently, a phase III study is investigating the role of venetoclax in combination with ibrutinib and obinutuzumab versus ibrutinib plus obinutuzumab in treatment-naïve young patients with CLL. This phase III trial builds from the efficacy of an earlier trial utilizing this combination, 116 and hopes to answer the question of whether this time-limited three-drug combination is effective versus continuous ibrutinib dosing in CLL patients aged younger than 70 years [ ClinicalTrials.gov identifier: NCT03701282 ]. Yet another study evaluating this combination is also underway, wherein each drug is given as a sequential therapy, beginning with ibrutinib as induction therapy, obinutuzumab as consolidation, and venetoclax as a 1-year maintenance [ ClinicalTrials.gov identifier: NCT03755947 ].

Immunological therapies

Checkpoint inhibitors

Nivolumab is an antibody against programmed-cell death protein 1 (PD-1), the blockade of which releases the ‘checkpoint inhibition’ of antitumor T-cell responses, allowing for T-cell-induced tumor killing. 117 Nivolumab in combination with ibrutinib in a high-risk [del(17p) and del(11q)] relapsed refractory CLL population led to an ORR of 61%. 118 Grade 3–4 immune-related toxicities included rash in 8% of patients and elevated alanine transaminase in 2%. 118 While this combination was well tolerated, the response rate was similar to single-agent ibrutinib in CLL. Therefore, it is unclear if the addition of nivolumab potentiates the clinical efficacy of ibrutinib. However, it has been noted that 65% of cases with Richter’s transformation responded to the combination of ibrutinib and nivolumab, demonstrating that this combination may be useful in this setting. 118 , 119

Likewise, pembrolizumab, another anti-PD-1-antibody has shown efficacy as a single agent in patients with Richter’s transformation. 120 However, no patients with CLL responded to pembrolizumab in this study. 120 A study investigating the combination of pembrolizumab with either idelalisib or ibrutinib is currently underway [ ClinicalTrials.gov identifier: NCT02332980 ].

Chimeric antigen receptor T cells

Modifying one’s own T cells to attack cancer is no longer science fiction. June and colleagues were the first to report the use of autologous chimeric antigen receptor (CAR)-modified T cells in a multirefractory CLL patient, who then achieved a CR. 121 This technology programs T cells to recognize CD19 on the surface of normal and malignant B cells and then selectively kill CD19-expressing cells. 121

Currently, similar CAR-T cells are being evaluated in multiple prospective trials. A phase I CD19 CAR-T trial enrolled very high risk and ibrutinib-refractory CLL patients. Most patients had either complex karyotype or del(17p) (23/24). 122 ORR was 71% (17/24), and for patients who were alive at the restaging time point, CR was 21% (4/19) and partial response was 53% (10/19). 122 Toxicities were similar to previous CAR-T studies, with cytokine release syndrome and neurotoxicity observed in 83% and 33%, respectively. In those who had a clinical response and underwent bone marrow biopsy, 88% (15/17) had no MRD by flow cytometry, and 58% (7/12) had no detectable disease by deep IGH sequencing. 122 This demonstrates that patients who respond to CAR-T therapy may be able to achieve a deep remission without MRD.

Other CAR therapies are also in development, such as ‘off the shelf’ allogeneic CAR-T-cell therapy, 123 and natural killer (NK) CAR-cell therapy, the latter of which is being actively developed for refractory CLL [ ClinicalTrials.gov identifier: NCT02727803 ].

Allogeneic transplantation

At this time, the only truly ‘curative’ treatment option for hematologic malignancy is allogeneic stem-cell transplantation, which potentially offers a graft versus leukemia effect, leading to a cure. This possible benefit is substantial, but the risk is also high. Given the option, many patients with TP53 abnormalities seek allogeneic transplantation. At 6 years post-allogeneic transplant, data from four large centers showed a PFS of 40–45% and OS of 50–60% in this patient population. 124 – 126 These were patients who had matching donors and were selected to be ‘fit’. Treatment-related mortality and severe graft versus host disease was reported in 16–25% and 50–55%, respectively. These results make it daunting to recommend allogeneic transplantation in many elderly patients, as risk will undoubtedly be worse for elderly patients who are under-represented in these clinical trials. However, for a young, fit, high-risk CLL patient with a perfectly matched donor, the opportunity of a potentially curative therapy may be worth the risks associated with stem-cell transplantation. Indeed, the evolving reality of targeted, orally available, and well-tolerated therapies is reason to pause prior to recommending transplant. However, this higher-risk option still represents a viable option while awaiting longer-term outcomes for patients treated with such targeted agents.

p53-targeted therapy

Indirect activation of p53 function by inhibition of MDM2 has been achieved preclinically with a small-molecule antagonist, Nutlin-3a. 127 However, the development of this agent has not been fully deployed in the clinical setting due to other exciting therapeutic options, deflating clinical interest in MDM2 as a pharmacologic target. Given that p53 mutations represent a significant mechanism driving poor outcomes in patients with CLL, therapies that restore wild-type p53 function may still be useful additions to the armamentarium of antileukemic agents. In fact, a recently opened phase III clinical trial [ ClinicalTrials.gov identifier: NCT03745716 ] is attempting to reactivate mutant p53 functions using APR-246 (formally known as PRIMA-1MET) in patients with refractory myelodysplastic syndrome. Successful results from this trial could provide impetus to explore the activity of APR-246 in patients with CLL harboring p53 mutations.

Resistance

In cases of CLL that develop resistance to BTK inhibitors ibrutinib or acalabrutinib, a substantially higher percentage appear to harbor del(17p). 128 , 129 Such an association suggests that being aware of resistance mechanisms in this population may be particularly prudent.

Restoration of BCR signaling and resistance to ibrutinib have been observed via mutations in BTK at Cys481, thus preventing ibrutinib from binding. 128 – 131 Activating mutations in the downstream substrate PLCγ2 leading to autonomous BCR signaling are also commonly observed. 128 – 131 In such cases, transition of therapy to venetoclax or a PI3Kδ inhibitor can suppresses these resistant clones. 128 , 129 , 132 Development of small molecules to specifically address these resistance mutations may also hold promise. LOXO305 is a reversible BTK inhibitor that retains activity against the most common BTK C481S alteration in vitro . 133 A phase I trial is underway with this agent [ ClinicalTrials.gov identifier: NCT03740529 ]. Likewise, ARQ 531 is a reversible BTK inhibitor with other kinase inhibitory activity that has been shown in preclinical studies to be effective against both BTK and PLCγ2 mutant clones. 134

Resistance to venetoclax can arise from a mutation in BCL2 at Gly101 or Asp103. Mutations at these residue can reduce the affinity of venetoclax for BCL2 substantially. These mutations have been associated with venetoclax resistance and impending relapse in patients with CLL. 135 , 136 Other antiapoptotic proteins besides BCL2, such as MCL1 or BCL-X L , may also be upregulated in CLL cells, leading to disease progression despite venetoclax administration. Importantly, inhibitors of BCR signaling (ibrutinib, idelalisib) and blockade of CD20 (obinutuzumab, rituximab) have demonstrated downregulation of MCL1, which sensitizes leukemic cells to BCL2 inhibition by venetoclax, lending further credence to the notion of combination therapy. 137

Pre-emptively understanding these mechanisms of resistance and their impact on the p53 pathway will be key to the sustained success of these agents in the clinic. Understanding the known mechanisms of resistance or anticipating the development of resistance may be useful for keeping patients in deep, durable remissions.

Summary

Patients with CLL have benefited greatly from the explosion of new treatment options. We are rapidly moving away from genotoxic drugs in the frontline setting, and the new reality is that patients with CLL are being treated with targeted small-molecule inhibitors, achieving greater responses and deeper remissions in the context of improved quality of life. Likewise, TP53 -abnormal populations have also seen vast improvements in their survival with these new combinations. Likewise, novel CAR-T, NK CARs, and allogeneic CAR therapies have the potential to provide the next steps forward in improving the quality and quantity of life for all patients with CLL.

We thank Mrs. Xiaorui Zhang for critically reviewing the manuscript.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of interest statement: The author(s) declare that there is no conflict of interest.

ORCID iDs: Marisa JL Aitken https://orcid.org/0000-0002-7937-531X

Sean M Post https://orcid.org/0000-0001-9225-200X

References

1 Jemal A Siegel R Ward E , et al Cancer statistics, 2007 . CA Cancer J Clin 2007 ; 57 : 43 – 66 . 17237035 10.3322/canjclin.57.1.43

2 National Cancer Institute Surveillance, Epidemiology, and the End Results Program . Cancer fact sheets: chronic lymphocytic leukemia (CLL) , https://seer.cancer.gov/statfacts/html/clyl.html ( accessed 1 July 2019 ).

3 Hallek M Cheson BD Catovsky D , et al iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL . Blood 2018 ; 131 : 2745 – 2760 . 29540348 10.1182/blood-2017-09-806398

4 Rozman C Montserrat E. Chronic lymphocytic leukemia . N Engl J Med 1995 ; 333 : 1052 – 1057 . 7675049 10.1056/NEJM199510193331606

5 Dohner H Stilgenbauer S Benner A , et al Genomic aberrations and survival in chronic lymphocytic leukemia . N Engl J Med 2000 ; 343 : 1910 – 1916 . 11136261 10.1056/NEJM200012283432602

6 Grever MR Lucas DM Dewald GW , et al Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US intergroup phase III trial E2997 . J Clin Oncol 2007 ; 25 : 799 – 804 . 17283363 10.1200/JCO.2006.08.3089

7 Catovsky D Richards S Matutes E , et al Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 trial): a randomised controlled trial . Lancet 2007 ; 370 : 230 – 239 . 17658394 10.1016/S0140-6736(07)61125-8

8 Hallek M Fischer K Fingerle-Rowson G , et al Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial . Lancet 2010 ; 376 : 1164 – 1174 . 20888994 10.1016/S0140-6736(10)61381-5

9 Stilgenbauer S Zenz T Winkler D , et al Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German chronic lymphocytic leukemia study group . J Clin Oncol 2009 ; 27 : 3994 – 4001 . 19597025 10.1200/JCO.2008.21.1128

10 Lane DP Cancer. p53, guardian of the genome . Nature 1992 ; 358 : 15 – 16 . 1614522 10.1038/358015a0

11 Malkin D Li FP Strong LC , et al Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms . Science 1990 ; 250 : 1233 – 1238 . 1978757 10.1126/science.1978757

12 Zenz T Eichhorst B Busch R , et al TP53 mutation and survival in chronic lymphocytic leukemia . J Clin Oncol 2010 ; 28 : 4473 – 4479 . 20697090 10.1200/JCO.2009.27.8762

13 Zenz T Krober A Scherer K , et al Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up . Blood 2008 ; 112 : 3322 – 3329 . 18689542 10.1182/blood-2008-04-154070

14 Rossi D Cerri M Deambrogi C , et al The prognostic value of TP53 mutations in chronic lymphocytic leukemia is independent of Del17p13: implications for overall survival and chemorefractoriness . Clin Cancer Res 2009 ; 15 : 995 – 1004 . 19188171 10.1158/1078-0432.CCR-08-1630

15 Momand J Zambetti GP Olson DC , et al The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation . Cell 1992 ; 69 : 1237 – 1245 . 1535557 10.1016/0092-8674(92)90644-r

16 Haupt Y Maya R Kazaz A , et al Mdm2 promotes the rapid degradation of p53 . Nature 1997 ; 387 : 296 – 299 . 9153395 10.1038/387296a0

17 Honda R Tanaka H Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 . FEBS Lett 1997 ; 420 : 25 – 27 . 9450543 10.1016/s0014-5793(97)01480-4

18 Kubbutat MH Jones SN Vousden KH. Regulation of p53 stability by Mdm2 . Nature 1997 ; 387 : 299 – 303 . 9153396 10.1038/387299a0

19 Abraham RT. PI 3-kinase related kinases: ‘big’ players in stress-induced signaling pathways . DNA Repair (Amst) 2004 ; 3 : 883 – 887 . 15279773 10.1016/j.dnarep.2004.04.002

20 Shieh SY Ikeda M Taya Y , et al DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2 . Cell 1997 ; 91 : 325 – 334 . 9363941 10.1016/s0092-8674(00)80416-x

21 Maya R Balass M Kim ST , et al ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage . Genes Dev 2001 ; 15 : 1067 – 1077 . 11331603 10.1101/gad.886901 PMC312683

22 Khosravi R Maya R Gottlieb T , et al Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage . Proc Natl Acad Sci U S A 1999 ; 96 : 14973 – 14977 . 10611322 10.1073/pnas.96.26.14973 PMC24757

23 Waldman T Kinzler KW Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells . Cancer Res 1995 ; 55 : 5187 – 5190 . 7585571

24 Miyashita T Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene . Cell 1995 ; 80 : 293 – 299 . 7834749 10.1016/0092-8674(95)90412-3

25 Nakano K Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53 . Mol Cell 2001 ; 7 : 683 – 694 . 11463392 10.1016/s1097-2765(01)00214-3

26 Oda E Ohki R Murasawa H , et al Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis . Science 2000 ; 288 : 1053 – 1058 . 10807576 10.1126/science.288.5468.1053

27 Miyashita T Harigai M Hanada M , et al Identification of a p53-dependent negative response element in the bcl-2 gene . Cancer Res 1994 ; 54 : 3131 – 3135 . 8205530

28 Miyashita T Krajewski S Krajewska M , et al Tumor suppressor p53 is a regulator of Bcl-2 and Bax gene expression in vitro and in vivo . Oncogene 1994 ; 9 : 1799 – 1805 . 8183579

29 Schuler M Bossy-Wetzel E Goldstein JC , et al p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release . J Biol Chem 2000 ; 275 : 7337 – 7342 . 10702305 10.1074/jbc.275.10.7337

30 Bouaoun L Sonkin D Ardin M , et al TP53 Variations in human cancers: new lessons from the IARC TP53 database and genomics data . Hum Mutat 2016 ; 37 : 865 – 876 . 27328919 10.1002/humu.23035

31 Donehower LA Harvey M Slagle BL , et al Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours . Nature 1992 ; 356 : 215 – 221 . 1552940 10.1038/356215a0

32 Jacks T Remington L Williams BO , et al Tumor spectrum analysis in p53-mutant mice . Curr Biol 1994 ; 4 : 1 – 7 . 7922305 10.1016/s0960-9822(00)00002-6

33 Lang GA Iwakuma T Suh YA , et al Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome . Cell 2004 ; 119 : 861 – 872 . 15607981 10.1016/j.cell.2004.11.006

34 Olive KP Tuveson DA Ruhe ZC , et al Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome . Cell 2004 ; 119 : 847 – 860 . 15607980 10.1016/j.cell.2004.11.004

35 Mantovani F Collavin L Del Sal G. Mutant p53 as a guardian of the cancer cell . Cell Death Differ 2019 ; 26 : 199 – 212 . 30538286 10.1038/s41418-018-0246-9 PMC6329812

36 Xu-Monette ZY Medeiros LJ Li Y , et al Dysfunction of the TP53 tumor suppressor gene in lymphoid malignancies . Blood 2012 ; 119 : 3668 – 3683 . 22275381 10.1182/blood-2011-11-366062 PMC3335376

37 Watanabe T Hotta T Ichikawa A , et al The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin . Blood 1994 ; 84 : 3158 – 3165 . 7949188

38 Post SM Pant V Abbas H , et al Prognostic impact of the MDM2SNP309 allele in leukemia and lymphoma . Oncotarget 2010 ; 1 : 168 – 174 . 21301048 10.18632/oncotarget.123 PMC3157715

39 Gryshchenko I Hofbauer S Stoecher M , et al MDM2 SNP309 is associated with poor outcome in B-cell chronic lymphocytic leukemia . J Clin Oncol 2008 ; 26 : 2252 – 2257 . 18467716 10.1200/JCO.2007.11.5212

40 Bo MD Secchiero P Degan M , et al MDM4 (MDMX) is overexpressed in chronic lymphocytic leukaemia (CLL) and marks a subset of p53wild-type CLL with a poor cytotoxic response to Nutlin-3 . Br J Haematol 2010 ; 150 : 237 – 239 . 20507307 10.1111/j.1365-2141.2010.08185.x

41 Jones SN Hancock AR Vogel H , et al Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis . Proc Natl Acad Sci U S A 1998 ; 95 : 15608 – 15612 . 9861017 10.1073/pnas.95.26.15608 PMC28091

42 Xiong S Pant V Suh YA , et al Spontaneous tumorigenesis in mice overexpressing the p53-negative regulator Mdm4 . Cancer Res 2010 ; 70 : 7148 – 7154 . 20736370 10.1158/0008-5472.CAN-10-1457 PMC2940952

43 Kamijo T Weber JD Zambetti G , et al Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2 . Proc Natl Acad Sci U S A 1998 ; 95 : 8292 – 8297 . 9653180 10.1073/pnas.95.14.8292 PMC20969

44 Zhang Y Xiong Y Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways . Cell 1998 ; 92 : 725 – 734 . 9529249 10.1016/s0092-8674(00)81401-4

45 Zhang Y Xiong Y. Control of p53 ubiquitination and nuclear export by MDM2 and ARF . Cell Growth Differ 2001 ; 12 : 175 – 186 . 11331246

46 Pinyol M Cobo F Bea S , et al p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin’s lymphomas . Blood 1998 ; 91 : 2977 – 2984 . 9531609

47 CLL Trialists’ Collaborative Group . Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials . J Natl Cancer Inst 1999 ; 91 : 861 – 868 . 10340906 10.1093/jnci/91.10.861

48 Langerbeins P Bahlo J Rhein C , et al The CLL12 trial protocol: a placebo-controlled double-blind phase III study of ibrutinib in the treatment of early-stage chronic lymphocytic leukemia patients with risk of early disease progression . Future Oncol 2015 ; 11 : 1895 – 1903 . 26161926 10.2217/fon.15.95

49 Langerbeins P Bahlo J Rhein C , et al Ibrutinib versus placebo in patients with asymptomatic, treatment-naïve early stage CLL: primary endpoint results of the phase 3 double-blind randomized CLL12 TRIAL . Hematol Oncol 2019 ; 37 : 38 – 40 .31187520

50 Di Antonio M McLuckie KI Balasubramanian S Reprogramming the mechanism of action of chlorambucil by coupling to a G-quadruplex ligand . J Am Chem Soc 2014 ; 136 : 5860 – 5863 . 24697838 10.1021/ja5014344 PMC4132976

51 Sawitsky A Rai KR Glidewell O , et al Comparison of daily versus intermittent chlorambucil and prednisone therapy in the treatment of patients with chronic lymphocytic leukemia . Blood 1977 ; 50 : 1049 – 1059 . 336116

52 Sylvan SE Asklid A Johansson H , et al First-line therapy in chronic lymphocytic leukemia: a Swedish nation-wide real-world study on 1053 consecutive patients treated between 2007 and 2013 . Haematologica 2019 ; 104 : 797 – 804 . 30467205 10.3324/haematol.2018.200204 PMC6442960

53 Al-Sawaf O Bahlo J Robrecht S , et al Outcome of patients aged 80 years or older treated for chronic lymphocytic leukaemia . Br J Haematol 2018 ; 183 : 727 – 735 . 30460980 10.1111/bjh.15604

54 Gonzalez D Martinez P Wade R , et al Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial . J Clin Oncol 2011 ; 29 : 2223 – 2229 . 21483000 10.1200/JCO.2010.32.0838

55 Chun HG Leyland-Jones B Cheson BD. Fludarabine phosphate: a synthetic purine antimetabolite with significant activity against lymphoid malignancies . J Clin Oncol 1991 ; 9 : 175 – 188 . 1702143 10.1200/JCO.1991.9.1.175

56 Rai KR Peterson BL Appelbaum FR , et al Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia . N Engl J Med 2000 ; 343 : 1750 – 1757 . 11114313 10.1056/NEJM200012143432402

57 Schnaiter A Paschka P Rossi M , et al NOTCH1, SF3B1, and TP53 mutations in fludarabine-refractory CLL patients treated with alemtuzumab: results from the CLL2H trial of the GCLLSG . Blood 2013 ; 122 : 1266 – 1270 . 23821658 10.1182/blood-2013-03-488197

58 Roue G Lopez-Guerra M Milpied P , et al Bendamustine is effective in p53-deficient B-cell neoplasms and requires oxidative stress and caspase-independent signaling . Clin Cancer Res 2008 ; 14 : 6907 – 6915 . 18980985 10.1158/1078-0432.CCR-08-0388

59 Leoni LM Bailey B Reifert J , et al Bendamustine (Treanda) displays a distinct pattern of cytotoxicity and unique mechanistic features compared with other alkylating agents . Clin Cancer Res 2008 ; 14 : 309 – 317 . 18172283 10.1158/1078-0432.CCR-07-1061

60 Fischer K Cramer P Busch R , et al Bendamustine in combination with rituximab for previously untreated patients with chronic lymphocytic leukemia: a multicenter phase II trial of the German chronic lymphocytic leukemia study group . J Clin Oncol 2012 ; 30 : 3209 – 3216 . 22869884 10.1200/JCO.2011.39.2688

61 Eichhorst B Fink AM Bahlo J , et al First-line chemoimmunotherapy with bendamustine and rituximab versus fludarabine, cyclophosphamide, and rituximab in patients with advanced chronic lymphocytic leukaemia (CLL10): an international, open-label, randomised, phase 3, non-inferiority trial . Lancet Oncol 2016 ; 17 : 928 – 942 . 27216274 10.1016/S1470-2045(16)30051-1

62 Riley JK Sliwkowski MX. CD20: a gene in search of a function . Semin Oncol 2000 ; 27 : 17 – 24 . 11225995

63 Weiner GJ. Rituximab: mechanism of action . Semin Hematol 2010 ; 47 : 115 – 123 . 20350658 10.1053/j.seminhematol.2010.01.011 PMC2848172

64 Mohammed R Milne A Kayani K , et al How the discovery of rituximab impacted the treatment of B-cell non-Hodgkin’s lymphomas . J Blood Med 2019 ; 10 : 71 – 84 . 30881167 10.2147/JBM.S190784 PMC6398399

65 Badoux XC Keating MJ Wang X , et al Fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy is highly effective treatment for relapsed patients with CLL . Blood 2011 ; 117 : 3016 – 3024 . 21245487 10.1182/blood-2010-08-304683 PMC4123386

66 Parikh SA Keating MJ O’Brien S , et al Frontline chemoimmunotherapy with fludarabine, cyclophosphamide, alemtuzumab, and rituximab for high-risk chronic lymphocytic leukemia . Blood 2011 ; 118 : 2062 – 2068 . 21750315 10.1182/blood-2011-01-329177 PMC4081295

67 Teeling JL French RR Cragg MS , et al Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas . Blood 2004 ; 104 : 1793 – 1800 . 15172969 10.1182/blood-2004-01-0039

68 Wierda WG Kipps TJ Mayer J , et al Ofatumumab as single-agent CD20 immunotherapy in fludarabine-refractory chronic lymphocytic leukemia . J Clin Oncol 2010 ; 28 : 1749 – 1755 . 20194866 10.1200/JCO.2009.25.3187 PMC4979101

69 Goede V Fischer K Busch R , et al Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions . N Engl J Med 2014 ; 370 : 1101 – 1110 . 24401022 10.1056/NEJMoa1313984

70 Brown JR O’Brien S Kingsley CD , et al Obinutuzumab plus fludarabine/cyclophosphamide or bendamustine in the initial therapy of CLL patients: the phase 1b GALTON trial . Blood 2015 ; 125 : 2779 – 2785 . 25769620 10.1182/blood-2014-12-613570 PMC4416529

71 Ferrajoli A O’Brien S Keating MJ. Alemtuzumab: a novel monoclonal antibody . Expert Opin Biol Ther 2001 ; 1 : 1059 – 1065 . 11728236 10.1517/14712598.1.6.1059

72 Keating MJ O’Brien S Kontoyiannis D , et al Results of first salvage therapy for patients refractory to a fludarabine regimen in chronic lymphocytic leukemia . Leuk Lymphoma 2002 ; 43 : 1755 – 1762 . 12685828 10.1080/1042819021000006547

73 Keating MJ Flinn I Jain V , et al Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study . Blood 2002 ; 99 : 3554 – 3561 . 11986207 10.1182/blood.v99.10.3554

74 Petlickovski A Laurenti L Li X , et al Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells . Blood 2005 ; 105 : 4820 – 4827 . 15728130 10.1182/blood-2004-07-2669

75 Stevenson FK Krysov S Davies AJ , et al B-cell receptor signaling in chronic lymphocytic leukemia . Blood 2011 ; 118 : 4313 – 4320 . 21816833 10.1182/blood-2011-06-338855

76 Kitada S Andersen J Akar S , et al Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses . Blood 1998 ; 91 : 3379 – 3389 . 9558396

77 Hanada M Delia D Aiello A , et al bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia . Blood 1993 ; 82 : 1820 – 1828 . 8104532

78 Kitada S Takayama S De Riel K , et al Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression . Antisense Res Dev 1994 ; 4 : 71 – 79 . 7950302 10.1089/ard.1994.4.71

79 Yang Q Modi P Newcomb T , et al Idelalisib: first-in-class PI3K delta inhibitor for the treatment of chronic lymphocytic leukemia, small lymphocytic leukemia, and follicular lymphoma . Clin Cancer Res 2015 ; 21 : 1537 – 1542 . 25670221 10.1158/1078-0432.CCR-14-2034 PMC4523214

80 Okkenhaug K Bilancio A Farjot G , et al Impaired B and T cell antigen receptor signaling in p110δ PI 3-kinase mutant mice . Science 2002 ; 297 : 1031 – 1034 . 12130661 10.1126/science.1073560

81 Clayton E Bardi G Bell SE , et al A crucial role for the p110δ subunit of phosphatidylinositol 3-kinase in B cell development and activation . J Exp Med 2002 ; 196 : 753 – 763 . 12235209 10.1084/jem.20020805 PMC2194055

82 Jou ST Carpino N Takahashi Y , et al Essential, nonredundant role for the phosphoinositide 3-kinase p110δ in signaling by the B-cell receptor complex . Mol Cell Biol 2002 ; 22 : 8580 – 8591 . 12446777 10.1128/MCB.22.24.8580-8591.2002 PMC139888

83 Lannutti BJ Meadows SA Herman SE , et al CAL-101, a p110δ selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability . Blood 2011 ; 117 : 591 – 594 . 20959606 10.1182/blood-2010-03-275305 PMC3694505

84 Longo PG Laurenti L Gobessi S , et al The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells . Blood 2008 ; 111 : 846 – 855 . 17928528 10.1182/blood-2007-05-089037

85 Rodriguez A Villuendas R Yanez L , et al Molecular heterogeneity in chronic lymphocytic leukemia is dependent on BCR signaling: clinical correlation . Leukemia 2007 ; 21 : 1984 – 1991 . 17611561 10.1038/sj.leu.2404831

86 Ogawara Y Kishishita S Obata T , et al Akt enhances Mdm2-mediated ubiquitination and degradation of p53 . J Biol Chem 2002 ; 277 : 21843 – 21850 . 11923280 10.1074/jbc.M109745200

87 Yang S Zhu Z Zhang X , et al Idelalisib induces PUMA-dependent apoptosis in colon cancer cells . Oncotarget 2017 ; 8 : 6102 – 6113 . 28008149 10.18632/oncotarget.14043 PMC5351616

88 Furman RR Sharman JP Coutre SE , et al Idelalisib and rituximab in relapsed chronic lymphocytic leukemia . N Engl J Med 2014 ; 370 : 997 – 1007 . 24450857 10.1056/NEJMoa1315226 PMC4161365

89 Sharman JP Coutre SE Furman RR , et al Final results of a randomized, phase III study of rituximab with or without idelalisib followed by open-label idelalisib in patients with relapsed chronic lymphocytic leukemia . J Clin Oncol 2019 ; 37 : 1391 – 1402 . 30995176 10.1200/JCO.18.01460 PMC10448866

90 De Weerdt I Koopmans SM Kater AP , et al Incidence and management of toxicity associated with ibrutinib and idelalisib: a practical approach . Haematologica 2017 ; 102 : 1629 – 1639 . 28775119 10.3324/haematol.2017.164103 PMC5622847

91 Lampson BL Kasar SN Matos TR , et al Idelalisib given front-line for treatment of chronic lymphocytic leukemia causes frequent immune-mediated hepatotoxicity . Blood 2016 ; 128 : 195 – 203 . 27247136 10.1182/blood-2016-03-707133 PMC4946200

92 Coutre SE Barrientos JC Brown JR , et al Management of adverse events associated with idelalisib treatment: expert panel opinion . Leuk Lymphoma 2015 ; 56 : 2779 – 2786 . 25726955 10.3109/10428194.2015.1022770 PMC4732460

93 Zelenetz AD Barrientos JC Brown JR , et al Idelalisib or placebo in combination with bendamustine and rituximab in patients with relapsed or refractory chronic lymphocytic leukaemia: interim results from a phase 3, randomised, double-blind, placebo-controlled trial . Lancet Oncol 2017 ; 18 : 297 – 311 . 28139405 10.1016/S1470-2045(16)30671-4 PMC5589180

94 Flinn IW Hillmen P Montillo M , et al The phase 3 DUO trial: duvelisib vs ofatumumab in relapsed and refractory CLL/SLL . Blood 2018 ; 132 : 2446 – 2455 . 30287523 10.1182/blood-2018-05-850461 PMC6284216

95 Burris HA III Flinn IW Patel MR , et al Umbralisib, a novel PI3Kδ and casein kinase-1epsilon inhibitor, in relapsed or refractory chronic lymphocytic leukaemia and lymphoma: an open-label, phase 1, dose-escalation, first-in-human study . Lancet Oncol 2018 ; 19 : 486 – 496 . 29475723 10.1016/S1470-2045(18)30082-2

96 Mato AR Schuster SJ Lamanna N , et al A phase 2 study to assess the safety and efficacy of umbralisib in patients with chronic lymphocytic leukemia (CLL) who are intolerant to prior BTK or PI3K delta inhibitor therapy . Hematol Oncol 2019 ; 37 : 88 – 89 .

97 Byrd JC Furman RR Coutre SE , et al Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia . N Engl J Med 2013 ; 369 : 32 – 42 . 23782158 10.1056/NEJMoa1215637 PMC3772525

98 Woyach JA Ruppert AS Heerema NA , et al Ibrutinib regimens versus chemoimmunotherapy in older patients with untreated CLL . N Engl J Med 2018 ; 379 : 2517 – 2528 . 30501481 10.1056/NEJMoa1812836 PMC6325637

99 O’Brien S Jones JA Coutre SE , et al Ibrutinib for patients with relapsed or refractory chronic lymphocytic leukaemia with 17p deletion (RESONATE-17): a phase 2, open-label, multicentre study . Lancet Oncol 2016 ; 17 : 1409 – 1418 . 27637985 10.1016/S1470-2045(16)30212-1

100 Moreno C Greil R Demirkan F , et al Ibrutinib plus obinutuzumab versus chlorambucil plus obinutuzumab in first-line treatment of chronic lymphocytic leukaemia (iLLUMINATE): a multicentre, randomised, open-label, phase 3 trial . Lancet Oncol 2019 ; 20 : 43 – 56 . 30522969 10.1016/S1470-2045(18)30788-5

101 O’Brien S Furman RR Coutre S , et al Single-agent ibrutinib in treatment-naive and relapsed/refractory chronic lymphocytic leukemia: a 5-year experience . Blood 2018 ; 131 : 1910 – 1919 . 29437592 10.1182/blood-2017-10-810044 PMC5921964

102 Lee HJ Gallardo M Ma H , et al p53-independent ibrutinib responses in an Emu-TCL1 mouse model demonstrates efficacy in high-risk CLL . Blood Cancer J 2016 ; 6 : e434 . 27284738 10.1038/bcj.2016.41 PMC5141356

103 Byrd JC Harrington B O’Brien S , et al Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia . N Engl J Med 2016 ; 374 : 323 – 332 . 26641137 10.1056/NEJMoa1509981 PMC4862586

104 Ghia P Pluta A Wach M , et al Acalabrutinib vs rituximab plus idelalisib (IdR) or bendamustine (BR) by investigator choice in relapsed/refractory (RR) chronic lymphocytic leukemia: phase 3 ASCEND study . Hematol Oncol 2019 ; 37 : 86 – 87 .

105 Awan FT Schuh A Brown JR , et al Acalabrutinib monotherapy in patients with chronic lymphocytic leukemia who are intolerant to ibrutinib . Blood Adv 2019 ; 3 : 1553 – 1562 . 31088809 10.1182/bloodadvances.2018030007 PMC6517672

106 Rogers KA Thompson PA Allan JN , et al Phase 2 study of acalabrutinib in ibrutinib (IBR)-intolerant patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) . J Clin Oncol 2019 ; 37 : 7530 – 7530 .

107 Tam CS Trotman J Opat S , et al Phase 1 study of the selective BTK inhibitor zanubrutinib in B-cell malignancies and safety and efficacy evaluation in CLL . Blood 2019 ; 134 : 851 – 859 . 31340982 10.1182/blood.2019001160 PMC6742923

108 Hillmen P Brown JR Byrd JC , et al ALPINE: phase III zanubrutinib (BGB-3111) versus ibrutinib in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) . J Clin Oncol 2019 ; 37 : TPS7572–TPS7572.

109 Hardwick JM Soane L. Multiple functions of BCL-2 family proteins . Cold Spring Harb Perspect Biol 2013 ; 5 . pii: a008722. 10.1101/cshperspect.a008722 PMC3552500 23378584

110 Anderson MA Deng J Seymour JF , et al The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism . Blood 2016 ; 127 : 3215 – 3224 . 27069256 10.1182/blood-2016-01-688796 PMC4920022

111 Roberts AW Davids MS Pagel JM , et al Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia . N Engl J Med 2016 ; 374 : 311 – 322 . 26639348 10.1056/NEJMoa1513257 PMC7107002

112 Fischer K Al-Sawaf O Bahlo J , et al Venetoclax and obinutuzumab in patients with CLL and coexisting conditions . N Engl J Med 2019 ; 380 : 2225 – 2236 . 31166681 10.1056/NEJMoa1815281

113 Seymour JF Kipps TJ Eichhorst B , et al Venetoclax-rituximab in relapsed or refractory chronic lymphocytic leukemia . N Engl J Med 2018 ; 378 : 1107 – 1120 . 29562156 10.1056/NEJMoa1713976

114 Kater AP Seymour JF Hillmen P , et al Fixed duration of venetoclax-rituximab in relapsed/refractory chronic lymphocytic leukemia eradicates minimal residual disease and prolongs survival: post-treatment follow-up of the MURANO phase III study . J Clin Oncol 2019 ; 37 : 269 – 277 . 30523712 10.1200/JCO.18.01580

115 Jain N Keating M Thompson P , et al Ibrutinib and venetoclax for first-line treatment of CLL . N Engl J Med 2019 ; 380 : 2095 – 2103 . 31141631 10.1056/NEJMoa1900574 PMC11827445

116 Rogers KA Huang Y Ruppert AS , et al Phase 2 study of combination obinutuzumab, ibrutinib, and venetoclax in treatment-naive and relapsed/refractory chronic lymphocytic leukemia . Blood 2018 ; 132 : 693 – 693 .

117 Iwai Y Ishida M Tanaka Y , et al Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade . Proc Natl Acad Sci U S A 2002 ; 99 : 12293 – 12297 . 12218188 10.1073/pnas.192461099 PMC129438

118 Younes A Brody J Carpio C , et al Safety and activity of ibrutinib in combination with nivolumab in patients with relapsed non-Hodgkin lymphoma or chronic lymphocytic leukaemia: a phase 1/2a study . Lancet Haematol 2019 ; 6 : e67 – e78 . 30642819 10.1016/S2352-3026(18)30217-5

119 Jain N Basu S Thompson PA , et al Nivolumab combined with ibrutinib for CLL and richter transformation: a phase II trial . Blood 2016 ; 128 : 59 .

120 Ding W LaPlant BR Call TG , et al Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL . Blood 2017 ; 129 : 3419 – 3427 . 28424162 10.1182/blood-2017-02-765685 PMC5492091

121 Porter DL Levine BL Kalos M , et al Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia . N Engl J Med 2011 ; 365 : 725 – 733 . 21830940 10.1056/NEJMoa1103849 PMC3387277

122 Turtle CJ Hay KA Hanafi LA , et al Durable molecular remissions in chronic lymphocytic leukemia treated with CD19-specific chimeric antigen receptor-modified T cells after failure of ibrutinib . J Clin Oncol 2017 ; 35 : 3010 – 3020 . 28715249 10.1200/JCO.2017.72.8519 PMC5590803

123 Sheridan C. Allogene and celularity move CAR-T therapy off the shelf . Nat Biotechnol 2018 ; 36 : 375 – 377 . 29734306 10.1038/nbt0518-375

124 Sorror ML Storer BE Sandmaier BM , et al Five-year follow-up of patients with advanced chronic lymphocytic leukemia treated with allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning . J Clinical Oncol 2008 ; 26 : 4912 – 4920 . 18794548 10.1200/JCO.2007.15.4757 PMC2652085

125 Dreger P Schnaiter A Zenz T , et al TP53, SF3B1, and NOTCH1 mutations and outcome of allotransplantation for chronic lymphocytic leukemia: six-year follow-up of the GCLLSG CLL3X trial . Blood 2013 ; 121 : 3284 – 3288 . 23435461 10.1182/blood-2012-11-469627

126 Khouri IF Bassett R Poindexter N , et al Nonmyeloablative allogeneic stem cell transplantation in relapsed/refractory chronic lymphocytic leukemia: long-term follow-up, prognostic factors, and effect of human leukocyte histocompatibility antigen subtype on outcome . Cancer 2011 ; 117 : 4679 – 4688 . 21455998 10.1002/cncr.26091 PMC4371600

127 Kojima K Konopleva M McQueen T , et al Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia . Blood 2006 ; 108 : 993 – 1000 . 16543464 10.1182/blood-2005-12-5148 PMC1895860

128 Kanagal-Shamanna R Jain P Patel KP , et al Targeted multigene deep sequencing of Bruton tyrosine kinase inhibitor-resistant chronic lymphocytic leukemia with disease progression and Richter transformation . Cancer 2019 ; 125 : 559 – 574 . 30508305 10.1002/cncr.31831

129 Ahn IE Underbayev C Albitar A , et al Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia . Blood 2017 ; 129 : 1469 – 1479 . 28049639 10.1182/blood-2016-06-719294 PMC5356450

130 Woyach JA Furman RR Liu TM , et al Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib . N Engl J Med 2014 ; 370 : 2286 – 2294 . 24869598 10.1056/NEJMoa1400029 PMC4144824

131 Burger JA Landau DA Taylor-Weiner A , et al Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition . Nat Commun 2016 ; 7 : 11589 . 27199251 10.1038/ncomms11589 PMC4876453

132 Jones JA Mato AR Wierda WG , et al Venetoclax for chronic lymphocytic leukaemia progressing after ibrutinib: an interim analysis of a multicentre, open-label, phase 2 trial . Lancet Oncol 2018 ; 19 : 65 – 75 . 29246803 10.1016/S1470-2045(17)30909-9 PMC6027999

133 Brandhuber B Gomez E Smith S , et al LOXO-305, a next generation reversible BTK inhibitor, for overcoming acquired resistance to irreversible BTK inhibitors . Clin Lymphoma Myeloma Leuk 2018 ; 18 : S216.

134 Reiff SD Mantel R Smith LL , et al The BTK inhibitor ARQ 531 targets ibrutinib-resistant CLL and richter transformation . Cancer Discov 2018 ; 8 : 1300 – 1315 . 30093506 10.1158/2159-8290.CD-17-1409 PMC6261467

135 Blombery P Anderson MA Gong JN , et al Acquisition of the recurrent Gly101Val mutation in BCL2 confers resistance to venetoclax in patients with progressive chronic lymphocytic leukemia . Cancer Discov 2019 ; 9 : 342 – 353 . 30514704 10.1158/2159-8290.CD-18-1119

136 Tausch E Close W Dolnik A , et al Venetoclax resistance and acquired BCL2 mutations in chronic lymphocytic leukemia . Haematologica 2019 ; 104 : e434–e437. 10.3324/haematol.2019.222588 PMC6717583 31004028

137 Bagacean C Tomuleasa C Tempescul A , et al Apoptotic resistance in chronic lymphocytic leukemia and therapeutic perspectives . Crit Rev Clin Lab Sci 2019 ; 56 : 321 – 332 . 30935269 10.1080/10408363.2019.1600468