Over the past 50 years, and before the introduction of TA, the activity of the chemotherapy agents comprising alkylating agents (chlorambucil, cyclophosphamide and bendamustine); nucleoside analogs (fludarabine, pentostatin and cladribine) and corticosteroids was remarkable in patients with CLL. At the beginning, chlorambucil monotherapy was the therapeutic “gold standard” for several decades, but later, fludarabine-based regimens took advantage due to their superior overall response rates (ORR) compared with the other treatment regimens containing alkylating agents or corticosteroids [192].

In the early 2000s, the addition of anti-CD20 antibodies to chemotherapy resulted in prolonged survival, and CIT regimens therefore became the gold standard therapy. The combination of fludarabine, cyclophosphamide and rituximab (FCR) [193,194] was commonly used for younger, fit patients; bendamustine combined with rituximab (BR) [195,196,197] was commonly used for unfit patients and chlorambucil with anti-CD20 antibodies was used for elderly patients with coexisting conditions [198,199]. One of the potential risks of anti-CD20 antibodies is the reactivation of hepatitis B. Thus, virus B serologic testing is mandatory in all patients before anti-CD20 treatment initiation, and prophylactic antiviral therapy must be initiated before treatment in cases with a risk of reactivation. Another worrying issue associated with CIT is the long-term risk of inducing secondary neoplasia, including myelodysplastic syndromes and acute myeloid leukemia [61].

In the last years, some randomized clinical trials improved the survival and showed better side effect profiles with the TA [200,201]. Nowadays, the use of chemoimmunotherapy is steadily declining. An exception could be the group of young fit patients with IGHV-M, who often stay in remission for more than 10 years after treatment with the FCR regimen. For such patients, FCR treatment remains an alternative to the TA until we have a longer follow-up on ibrutinib-treated patients [61].

Ibrutinib is an oral small molecule acting as a Bruton tyrosine kinase inhibitor (BTKi). This drug is widely used nowadays not only as a frontline treatment but, also, in the relapse setting [113,202]. This is supported by the very satisfactory results recently shown in the phase 3 clinical trials RESONATE [203] and RESONATE-2 [204]. Even though, in both trials, the control arm was not the best “standard of care”, their results were impressive, showing a high ORR and survival benefit in the ibrutinib arm, with a follow-up of more than 5 years, for all the CLL subgroups. The first results from RESONATE showed the excellent efficacy of ibrutinib in refractory/relapse (R/R) CLL patients, leading to Food and Drug Administration (FDA) approval in 2014 [205]. The second clinical trial experimented the use of ibrutinib as a frontline therapy [204]. More recently, ibrutinib was compared to CIT in treatment-naive CLL patients, questioning the need for CIT even in the subgroup of young, low-risk patients. The combination of ibrutinib–rituximab (IR) was superior to FCR in terms of the PFS and OS in ECOG-ACRIN E1912. This benefit was observed for all the analyzed subgroups, with the exception of IGHV-M patients, in which both treatments achieved similar results, and a long follow-up is required to determine the best option for this population [201]. For patients not able to tolerate FCR, the ALLIANCE trial compared BR to ibrutinib +/− rituximab. Patients receiving ibrutinib showed a longer PFS than patients treated with BR. Benefits in the OS have not been observed to date, with a median follow-up of 38 months. Furthermore, rituximab did not improve the PFS compared to patients treated with ibrutinib monotherapy [200].

Ibrutinib is not free from adverse events, with the most frequent being mild diarrhea, fatigue, nausea, bruising and arthralgia, while the most severe and less common are infections, atrial fibrillation, hypertension and ventricular arrhythmia [210]. Additionally, the data from real-life studies show that the major cause of discontinuation is off-target toxicity rather than progression [211]. Probably, a better selection of patients with cardiovascular comorbidities or at a high risk of bleeding or infection can optimize this discontinuation rate. On the other hand, potential benefits of ibrutinib include a modulating effect on the immune system [212].

Currently, second-generation BTKi are under investigation. These inhibitors join more selectively to their therapeutic target, improving their toxicity profile due to less frequency of the off-target events. Of them, acalabrutinib is the most mature, as the FDA has recently approved it for CLL patients (first-line and relapse) based on last year’s results of the ELEVATE-TN [206] and ASCEND [207] phase 3 clinical trials. Both trials demonstrated superiority in the acalabrutinib arms, with a good safety profile, as shown in Table 2. Moreover, the addition of obinutuzumab to acalabrutinib could provide a better PFS than acalabrutinib monotherapy in therapy-naive CLL patients but neutropenia in 30% of patients [206]. Zanubrutinib or tirabrutinib are the other second-generation BKTi under clinical development. Both have demonstrated encouraging activity in CLL patients, with a low incidence of off-target toxicity in their phase 1 and 2 studies [213,214]. Specifically designed to overcome the acquired resistance to ibrutinib, a new family of reversible BTKi emerged in 2020. These agents are now in the early phases of research and are soon to demonstrate their applicability in real life. Among them, fenebrutinib, LOXO 305 and ARQ 531 are under active clinical investigation nowadays [215].

Venetoclax is an oral BCL-2 inhibitor highly active in patients with CLL. The clinical development of this drug has lagged behind that of ibrutinib, although its effectiveness seems just as promising. The first clinical trials of venetoclax in patients with R/R CLL showed high response rates in terms of the PFS and ORR across all subgroups of CLL patients [216,217]. Based on the CLL-14 and MURANO phase 3 trials (Table 2) [208,209,218,219], venetoclax in combination with anti-CD20 has been recently approved for frontline treatment and treatment for relapsed CLL. As opposed to BCR pathway inhibitors, venetoclax induces deep remissions with high rates of MRD that allow treatment discontinuation. To date, venetoclax plus obinutuzumab has yielded the highest MRD-negative response rate in a randomized trial so far [220]. An extended follow-up of the CLL-14 and MURANO trials has recently been published, confirming the notorious clinical benefit of the combinations with venetoclax and demonstrating an OS benefit for R/R patients treated with venetoclax–rituximab. The rates of MRD negativity were also significantly higher in the venetoclax arm of both trials [208,218].

Venetoclax requires special measures (initial ramp-up escalation dose, vigorous hydration and laboratory test monitoring) to mitigate the risk of tumoral lysis syndrome (TLS) observed in the first clinical studies. Taking into account the aforementioned factors, TLS is not a big concern and has been reported in a low proportion of patients. In contrast, the most frequent grade ≥3 adverse event is neutropenia detected in around 50–60% of the patients, although not followed by a higher risk of infection [208,218].

Idelalisib is the first-in-class phosphatidyl-inositol 3-kinase inhibitor (PI3Ki) used in R/R CLL patients. Its clinical development was contemporary to ibrutinib, and it has been demonstrated to be an active oral small molecule, preferably used in combination with rituximab. This was shown in a phase 3 study that randomized 220 patients to receive rituximab plus idelalisib or a placebo. Patients receiving idelalisib significantly improved their PFS (19 vs. 6 months) and their OS (41 vs. 35 months), despite an extensive cross-over [221], and achieved a high rate of ORR. However, these benefits seem inferior to those obtained with BTKi, as was recently confirmed in the ASCEND clinical trial [207].

Toxicity has limited the use of idelalisib in real life, with a high rate of infectious (pneumonia) and autoimmune side effects (colitis, pneumonitis and hepatitis). Duvelisib is another PI3Ki granted by the FDA in 2018 for the treatment of R/R CLL patients after at least two prior therapies. It has also been demonstrated to be active in CLL, but, again, toxicity might limit its widespread use. Umbralisib is a next-generation PI3Ki with a much better toxicity profile, as it has been related to fewer immune-mediated toxicities or severe opportunistic infections to date [222]. Different clinical trials of umbralisib alone or in combination are ongoing and will help to elucidate its role in this rapidly changing treatment era.

The surface antigen CD20 is the target of antibodies such as rituximab, ofatumumab and obinutuzumab, which are currently approved for CLL. These antibodies are commonly administered in combination with chemotherapy or targeted therapies.

Recently, advances in monoclonal antibody technology have resulted in the development of new antibodies with improved therapeutic effectiveness. Ublituximab stands out, a next-generation CD20 antibody with encouraging results, especially in combination with the targeted molecules [222] or other monoclonal antibodies such as cirmtuzumab (anti-ROR 1), MOR00208 (anti-CD19) or otlertuzumab (anti-CD37). Less advanced are the bispecific antibodies and immunomodulatory antibodies [215].

Alemtuzumab, as anti-CD52 monoclonal antibody, is approved for the treatment of CLL. It was indicated, before the TA “era”, especially for patients with del(17p)/TP53 mutations. However, the use of alemtuzumab is exceptional today due to the severe immunosuppression and the high rates of infectious complications associated with this drug [223]. Moxetumomab pasudotox, an antibody–drug conjugate targeting CD22 and delivering a cytotoxic agent simultaneously, has also been unsuccessful in treating CLL, unlike hairy cell leukemia. This is explained by the lower expression of CD22 in CLL lymphocytes [224].

TA have changed the treatment landscape of CLL. Combinations of these targeted treatments with CIT, CD20 monoclonal antibodies and between them is what the immediate future holds. This approach aims to limit the toxicity, cost and resistance and achieve profound responses with MRD that can lead to the potential curation of the disease and treatment discontinuation. In particular, existing evidence indicates that anti-CD20 plays a synergistic role when used in combination with venetoclax. The combination of second-generation anti-CD20, such as obinutuzumab with BTKi, also appears to be beneficial [196,218,220,225].

Regarding the combinations between CIT and TA, probably the most interesting studies are those including young, fit, treatment naïve patients with mutated IGHV. Some of them have demonstrated very high rates of negative MRD, allowing the discontinuation of TA. Ultimately, combinations between TA with or without the addition of anti-CD20 have shown preliminary promising outcomes, with high rates of MRD negativity making possible treatment discontinuation as well. Nowadays, countless clinical trials are ongoing on this field.

Moreover, three ongoing, independent phase 3 trials stand out (ECOG-ACRIN EA9161 for young patients, ALLIANCE A041702 for patients >70 years old and CLL-17 (fit and unfit patients)), exploring different combinations with venetoclax, ibrutinib and obinutuzumab that allow treatment disruption in some of their arms. The results of these studies will probably again change routine practices in the near future.

Allogeneic stem cell transplantation (allo-TPH) is a potentially curative approach to CLL patients. Years ago, it was indicated in patients with poor prognostic factors (early relapses, refractory to fludarabine or harboring TP53 abnormalities). TA have changed the natural history of CLL, and therefore, the role of allo-TPH in this new era is less clear. A recently published retrospective study reported the outcome of 65 patients undergoing allo-TPH after at least one TA, pointing out that it is a viable long-term disease control strategy. In this study, the investigators observed that PFS was predicted by the hematopoietic cell transplantation-specific comorbidity index. No differences were observed among the patients receiving previous TA (one or two ibrutinib/venetoclax) or TA and CIT as the previous treatment [235]. Currently, most guidelines recommend it for patients with high-risk CLL that have relapsed or are refractory to at least one TA or in cases of clonally related Richter transformation with a response to chemotherapy [202,236]. However, some questions, such as the optimal timing of the procedure, remain unanswered.

CLL was a pioneering disease in which chimeric antigenic receptor T (CAR-T) cells targeting CD19 were tested [237], but the ORRs were not as good as those observed in other diseases, and the estimated PFS at 18 months was around 28% [238]. In order to optimize its applicability in CLL, different strategies are under investigation, such as those using ibrutinib concurrently with CD19 CAR T cells [239]. With this approach, the ORR and PFS were improved, and a better toxicity profile was observed after one year of follow-up. Even so, these results need more robustness to be adopted in clinical practice. Another option under study is the use of modified cord blood natural killer cells to express anti-CD19 CAR [240].

While ibrutinib is an effective therapy leading to durable responses, some patients acquire resistance and relapse [242]. In 2014, a study using whole-exome sequencing discovered acquired mutations within the BTK gene in CLL patients relapsing on ibrutinib [243]. Further studies confirmed the presence of a BTK mutation in the CLL patients relapsing on ibrutinib [115,244,245], C481S being the most common mutation at the position of the binding site of the drug [246,247]. BTK mutations can be explained by the mechanism of action of ibrutinib, which binds to BTK with an irreversible covalent bond at position C481S. From there, ibrutinib inhibits the proliferative and antiapoptotic signals that are abnormally stimulated in CLL cells through the NF-κB pathway downstream a wide variety of signal transducers, including PLCG2, SYK or LYN, among others [248].

The second-most frequent mutations found in CLL patients who fail on ibrutinib treatment are PLCG2 mutations [249]. The PLCG2 gene encodes Cγ2, the protein immediately downstream of BTK, and its mutations mostly have an activating effect, resulting in continuous BCR signaling independent of the BTK activity [243,249].

The acquired mutations in these genes have been detected in 80% of patients with ibrutinib failure and CLL progression. Resistance usually develops between the second and fourth year of ibrutinib treatment, but BTK and PLCG2 mutations might be detected at low allelic frequencies up to 9–15 months before CLL progression [115,244]. In contrast to CLL progression on ibrutinib, which tends to occur later in therapy (after 12 months of attaining a response), Richter transformations mostly occur during the first 1 to 2 years of treatment [250,251].

On the other hand, there are approximately 20% of patients in whom BTK and PLCG2 mutations cannot be identified. For them, alternative mechanisms of resistance such as 8p deletion or additional driver mutations have been described and are shown in Figure 4 [252].

Intrinsic resistance to ibrutinib is extremely rare and, conversely, has not been studied in depth. Three independent studies analyzed pretreatment samples from patients who relapsed on ibrutinib treatment and failed to find mutations at that moment. In patients that relapse early (first fifteen months), it is necessary to rule out a transformation into a high-grade lymphoma [242,253].

Different strategies have been suggested to overcome ibrutinib resistance, highlighting the use of TA targeting other pathways such as PI3K or BCL-2 or the use of reversible BTK inhibitors [253].

The resistance mechanisms of venetoclax are not as well-defined as those occurring after ibrutinib failure. This could be due not only to the later development of the drug but, also, to the implication of different independent molecular mechanisms. Similar to what happens with BTK mutations, a mutation at the G101V in BCL2 has been implicated in the reduction of venetoclax binding to BCL2. This mutation was found in almost half of patients that progressed under venetoclax in a recent study of a small cohort of cases in 2019. Another mutation in BCL2, D103Y, has been also associated with venetoclax [254]. This and other mutations could coexist in same patients but as independent clones with different growth dynamics [254,255]. BCL2 mutations were identified several months prior to clinical relapse (~25) [256] (Table 4).

Besides BCL2 point mutations, other candidate resistance-associated aberrations have been reported, including mutations in the antiproliferative BTG1 gene, aberrations of CDKN2A/B, the overexpression of MCL1 and BCL-XL (pro-survival proteins) and the amplification of AMP-1, which can affect the OXPHOS pathway in mitochondria [155,256,257].