Background information upon the three studies (3-5 pages) Single Space Topic (Chronic lymphocytic leukemia (CLL)) ? ? ? ? ? Introduction to Chronic lymphocytic leukemia (CLL) Clinical features of Chronic lymphocytic leukemia (CLL) Diagnostic features/markers of Chronic lymphocytic leukemia (CLL) Uniqueness of the disease or differential diagnosis What is current treatment of Chronic lymphocytic leukemia (CLL)? Make a table: J. article/title Goal of the study Methods used Important findings conclusions Article 1 Article 2 Article 3 This gives a global view of the summary from the articles and helps to organize the paper: Objective: What is the main goal or objective (when you read the articles you choose, at the end of introduction, the authors outline their goal or objective. You can paraphrase or slightly modify and use as your objective STUDY • • Choose 3 articles and write as study 1, study 2 and study3 Briefly write introduction:1-3 paragraphs from EACH article STUDY 1: • • • Methods used in the article. Briefly mention results. Include PICTURES AND/OR TABLES IN SEPARATE PAGES Note: write the title of figure on table at the top, also number the figure or table write the table or figure Below the figure or table add: Write a brief description of method Write the summary or outcome of this figure or table You can directly transport this to PowerPoint then would be easy for you to present. Do the same for study 2, Study 3. STUDY 2: • • • Methods used in the article. Briefly mention results Include PICTURES AND/OR TABLES IN SEPARATE PAGES Note: Write the title of figure on table at the top; also number the figure or table Write the table or figure. Write below the figure or table, and add: Write a brief description of method Write the summary or outcome of this figure or table You can directly transport this to PowerPoint and becomes easy for you to present. Do the same for Study 3. STUDY 3: • • • Methods used in the article. Briefly mention results Include PICTURES AND/OR TABLES IN SEPARATE PAGES Note: write the title of figure on table at the top, also number the figure or table write the table or figure Below the figure or table add: Write a brief description of method Write the summary or outcome of this figure or table You can directly transport this to PowerPoint then would be easy for you to present. Finally: • • DISCUSSION 3-5 PAGES REFERENCES Easy way to do references: when you are writing the introduction/methods/discussion. (Include the first name and last name of the first author et al, Journal name, Page #s, year) Example John Deen et al., J. Immunol., p133-139, 2021). Do this to every reference in your paper. Once you finish the paper and finalized. Final step is: 1. 2. 3. 4. open “pubmed” from google. Copy the reference (as example above). Pubmed gives you full title of the article, all the references, and other information. Copy this and paste in your reference section Current Oncology Reports (2020) 22: 36 https://doi.org/10.1007/s11912-020-0893-0 LEUKEMIA (A AGUAYO, SECTION EDITOR) Chronic Lymphocytic Leukaemia in 2020: the Future Has Arrived Kate Milne 1,2 & Beattie Sturrock 1,2 & Timothy Chevassut 1,2 Published online: 14 March 2020 # The Author(s) 2020 Abstract Purpose of Review Chronic lymphocytic leukaemia is now recognised as a heterogenous disease with a variety of clinical outcomes. Here we summarise the way it is currently stratified according to genetic risk and patient characteristics and the treatment approaches used for these different subgroups. Recent Findings Certain patients appear to sustain MRD negativity after combination chemoimmunotherapy, leading to the suggestion that their CLL may be cured. However, 17p-deleted, p53-mutated or IGHV-UM subgroups are generally resistant to FCR, and much better responses are seen with ibrutinib and venetoclax, frequently inducing MRD negativity that hopefully will be translated into durable remissions. Summary Small molecule inhibitors have already revolutionised CLL treatment. Going forward, we anticipate their use in the majority of patients, early after diagnosis and with curative intent. Keywords Chronic lymphocytic leukaemia . Chemoimmunotherapy . Ibrutinib . Venetoclax . Future Introduction Chronic lymphocytic leukaemia (CLL) is the commonest leukaemia in the world, with 4.9 new diagnosis per 100,000 per year in the UK and USA. The malignant clonal proliferation and accumulation of mature B-lymphocytes is predominantly identified in older patients with a median age of 74 at diagnosis [1, 2]. The majority of patients are monitored with a ‘watch and wait approach’ until the balance of risks and benefits favours treatment initiation. In some cases, treatment may never be needed whilst in others, the disease is more aggressive with rapid progression and death from disease-related causes a few years after diagnosis [3]. This disparity in This article is part of the Topical Collection on Leukemia outcome highlights the heterogeneity of CLL and the importance of risk stratification to guide treatment decisions. unlike, its myeloid counterpart—chronic myeloid leukaemia—a pathognomonic driving mutation, BCR-ABL, has not been identified, and this slightly delayed the development of targeted therapies. However, over the last two decades, a dramatic increase in our understanding of the pathogenesis of the disease has led to the development of small molecule inhibitors for CLL targeting the B cell receptor pathway and the apoptotic regulator BCL2. Some of these newer therapies appear to be so effective; they may provide a curative option for patients, previously treatment aimed to establish disease control. In this review, we will briefly discuss recent advances in our understanding of the molecular pathology of CLL and then, in more detail, the way that CLL is managed in the UK. * Kate Milne kate.milne5@nhs.net Beattie Sturrock Beattie.sturrock@nhs.net Timothy Chevassut T.Chevassut@bsms.ac.uk 1 Brighton and Sussex Medical School, University of Sussex, Brighton BN1 9PS, UK 2 Royal Sussex County Hospital, Eastern Road, Brighton BN2 5BE, UK New Developments in the Understanding of the Pathogenies of CLL Our understanding of the genetics of CLL and the implications on patient outcome began with the publication by Doner et al. in 2000 which identified four recurrent genetic lesions by fluorescence in situ hybridisation (FISH) and showing that their presence predicted disease prognosis. It was found that 36 Page 2 of 9 a lesion at 17p13 and 11q23 indicated a poor prognosis compared with 13q14 and 12q; this formed the basis of the FISH probe set used routinely during CLL diagnostics [4]. The utility of FISH is limited by the need to identify particular region to probe and by the number of probes that can be used at one time. Unlike FISH, G-banding karyotyping which can be applied to all the chromosomes simultaneously, required cells to be in metaphase, various compounds have been used to raise the yield of metaphase cells in CLL—the combination of DSp30/IL2 has shown to do so without inducing artefactual chromosomal aberrations [5, 6]. Using this technique, it has been demonstrated 16–19% of patients have a complex karyotype (3 > abnormalities) that this predicts poor outcome independent of P53 status, and the presence of 5 of more is associated with a P53 mutations and perhaps due to this association, a more aggressive course [7]. These techniques have been replaced in research by chromosomal micro arrays and single nucleotide polymorphisms arrays which do not require the cells to be in the cell cycle and whilst the concordance of these approaches is reported in small studies, their incorporation into large trials is awaited [8]. CLL patients can be categorised into two groups depending on the mutational status, established by PCR or next generation sequencing, of the variable region immunoglobulin heavy chain (IGHV) when compared with germline sequence. During the process of VDJ rearrangement and somatic hypermutation of B cells occurs within the germinal centres generating receptors capable of recognising an extensive range of antigens. IGHV-mutated (IGHV-M) CLL, with a mutation status of > 2%, compared with germline sequence as an immunophenotype and gene expression profile similar to that of post GC CD27+, T cell dependent memory B cells. Whilst IGHV-unmutated (IGHV-UM) CLL cells adopted a phenotype gene expression profile and epigenome that resemble a GC-naïve CD27− B cell [9, 10]. This difference is reinforced when comparing the BCRs of the two subtypes with IGHVUM carrying low affinity, poly-reactive and self-reactive BCR, and IGHV-M has higher affinity, olio or mono reactive receptors [11]. This may contribute to the poorer prognosis seen with IGHV-UM CLL [12, 13]. Next generation sequencing confirmed that the mutational rate in CLL is similar to that of other haematological cancers and lower than solid malignancy, with each patients having a small number of recurrently mutated driver genes—unsurprisingly, many of the mutated genes identified in these studies are involved in cell replication, DNA repair apoptosis and signalling [14–17]. The subtypes, the 4 chromosomal abnormalities discussed earlier have also been shown to be associated with different recurrent gene mutations, similarly to IGHV status, which may explain the heterogeneity in there clinical course. For example, 17p13 deletion occurs with mutation of the remaining p53 resulting in homozygous inactivity and SF3B1, a protein involved in the regulation of the spliceosome with 11q Curr Oncol Rep (2020) 22: 36 deletion [18, 19]. In these studies, a quarter of patients had a mutation in a gene involved in RNA splicing or repair—a possible avenue for new drug development [20]. As well as identification of mutated genes in CLL, the mechanisms of gene expression regulation have also been studied. It is known that BCL2 an antiapoptotic protein is overexpressed in CLL and that its expression increases with chemotherapy [21–23]. BCL2 combines with BH3 and binds to BIM, preventing BIM triggering apoptosis [24]. The recurrent deletion of 13q14 is seen in 50% of de novo CLL; it has been shown that this deletion results in the loss of expression of micro RNA (miRNA) 15-A/16-1 [25, 26]. As miRNA 15A and 16-1 normal interfere in the transcription of BCL2, their loss contributes to its overexpression in CLL [27]. Interaction with other cells in the microenvironment is crucial to the survival and replication of CLL cells, as shown by the rapid apoptosis of CLL cells in vitro and the reduction in this when co-cultured with non-tumoral bystander cells [11]. The lymph nodes can be the key site of CLL proliferation, with higher activation of the NFKb and the BCR signalling pathways crucial for this process than the bone marrow of peripheral blood [28], whilst the bone marrow creates a protective niches preventing spontaneous and drug-induced apoptosis cells [29, 30]. Interruption of these interactions may force the cells into the peripheral circulation, therefore increasing their susceptibility to drug-induced chemotherapy. Treatment of Chronic Lymphocytic Leukaemia in 2019 Most patients with CLL present with an incidental finding of a lymphocytosis, they may have palpable lymphadenopathy or organomegaly, bone marrow involvement can lead to anaemia and thrombocytopenia. Occasionally, patients may have the constitutional B symptoms with an unexplained fever of over 38°, weight loss of > 10% in less than 6 months and night sweats. A complication of CLL such as a high-grade disease transformation, an autoimmune disease or a severe infection may lead to its diagnosis [31]. The following diagnostic criteria are stipulated by the international workshop of CLL (iwCLL) and reiterated in the WHO classification of lymphoid neoplasms. The lymphocytes count in the peripheral blood must be greater than 5 × 10 × 9, persistent for 3 months, and clonality of this population must be shown by flow cytometry for light chain restriction, CD5, CD23, CD79b and surface immunoglobulin expression with low levels of CD20. The presence of smudge/ smear cells, an artefact of blood film production, is a typical finding in CLL [32]. If the WCC is less than 5 × 10/L with no other signs of lymphoproliferative disorder, this constitutes monoclonal B cell lymphocytosis which is thought as a precursor to CLL, with a rate of progression of 1% per year [33]. Curr Oncol Rep (2020) 22: 36 Identification of a clonal population of mature B cells within the lymph nodes or extranodal tissues without a peripheral blood lymphocytosis is referred to as a small lymphocytic lymphoma [34]. Fluorescence in situ hybridisation is used to identify chromosomal rearrangements which can differentiate CLL from other conditions such as mantle cell lymphoma as well as helping with disease stratification. When to Initiate Treatment In addition to diagnostic criteria, the iwCLL guidelines dictate when to initiate treatment, based on patients’ symptoms, full blood counts and physical examination. The presence of constitutional symptoms, progressive lymphocytosis, a doubling time of less than 6 months, an Hb of less than 100 g/L or a platelet count of less than 100 x 109/L as well as progressive or symptomatic bulky lymphadenopathy/organomegaly or treatment-resistant autoimmune thrombocytopaenia or anaemia are indications to start treatment [32]. In some circumstances, treatment may be initiated at diagnosis, but it is much more common for patient to be monitored for signs of increasing disease activity often over many years. Patient Risk Stratification Similar to the management of most malignancy, the most suitable treatment for a patient with CLL is selected based on genetic features of the disease itself—the presence of a P53 mutation or 17p deletion and the mutational state of the IGHV—and patient factors [35]. The algorithm for treatment selection followed by haematologists in the UK is shown in Fig. 1. Disruption of P53, a tumour suppressor crucial in many cancers, is known to result in very poor response to combination immunochemotherapy, and therefore, patients are treated with molecular therapies first line [18]. If the IGHV is unmutated, compared with the germline sequence, this confers a poor survival risk when compared with patient with a mutated IGHV [12, 13]. The co-morbidities and performance status of individual patients are crucial in setting treatment goals and treatment selection. Treatment success or disease progression is determined on similar criteria to treatment initiation—repeat CT scans are not recommended out with clinical trials. The identification of minimal residual disease (MRD) on peripheral blood or bone marrow aspirate by flow cytometry, with a sensitivity of 0.01%, is becoming increasingly important as MRD negativity after chemoimmunotherapy is associated with prolonged progression-free and overall survival—in the future, early evidence of MRD negativity may allow for shorter treatment regimens [36, 37]. Page 3 of 9 36 Upon treatment failure on combination chemoimmunotherapy or ibrutinib, there are now several available options for relapsed disease including the PI3K inhibitor idelalisib and the BCL2 inhibitor Venetoclax, also shown in Fig. 1. Combination Immunochemotherapy—Fludarabine, Cyclophosphamide, Rituximab (FCR) and Other CD20 Antibodies Young fit patients, with a creatine clearance of greater than 70 and a comorbidity scale of 6 or less without P53 mutations and with mutated IGHV, are treated with combination chemoimmunotherapy, fludarabine, cyclophosphamide and rituximab (FCR) [38, 39]. Prior to the introduction of immunotherapy chlorambucil (CLB), an alkylating agent or fludarabine, a purine analogue monotherapy was the mainstay of treatment—the combination of these agents did not improve response rates but did carry much higher haematological toxicity [40]. The addition of cyclophosphamide to fludarabine (FC) led to an improved progression-free survival (PFS) and overall survival (OS) without an increase in serious adverse events compared with fludarabine monotherapy [41]. The approval of rituximab—a monoclonal antibody targeting CD20, a glycosylated cell surface protein expressed on mature B cells—provided a treatment option for many B cell malignancies. In the CLL8 trial, patients were randomised to receive either FC or FCR, which gave a response rate of 85 and 92% respectively. Particular subgroups of patients appear to have particularly good long-term outcomes, those with a mutated IGVH, del(13q), trisomy 12 or del(11q) or MRD negative remission [42, 43•]. The durability of these remissions led to the suggestion that FCR may be curative for some patients. There is emerging evidence that reduced doses or 3 rather than 6 cycles result in non-inferior PFS and OS with a lower burden of cumulative toxicity [36]. Whilst FCR is known to result in longer PFS compared with bendamustine and rituximab (BR) in younger patients, the benefit was not seen in patients over 65—and a lower rate of serious infections were seen in the BR cohort. Therefore, in patients over 65 who do not qualify for ibrutinib BR tended to be favoured, unless there is a contraindication to bendamustine when chlorambucil rituximab could be used accepting that this had a poorer response rate [44, 45]. The CLL11 trial looked specifically at the treatment of older patients with co-morbidities and the impact of a new anti CD20 monoclonal antibody, obinutuzumab. Treatment with obinutuzumab-CLB compared with R-CLB and CLB monthotherapy increased response rates and prolonged PFS (median PFS, 26.7 months with obinutuzumab-CLB vs 11.1 months with CLB alone; 16.3 months with R-CLB) 36 Page 4 of 9 Curr Oncol Rep (2020) 22: 36 Fig. 1 Flow chart showing the decision process and treatment options in management of previously untreated CLL that meets the iwCLL criteria for treatment and in relapsed or refractory disease Untreated CLL patient who meets the criteria for therapy Assess TP53 status Old and/or frail TP53 mutated IGHV not mutated Assess IGHV mutation status IGHV mutated Old and/or frail Young and fit Young and fit Obinutuzumab +/chlorambucil Or Ibrutinib Ibrutinib Or Obinatuzumab Ibrutinib Or FCR(BR if >65years) FCR(BR if >65 years) Ibrutinib Relapsed or refractory CLL Restaging: bone marrow aspirate and biopsy, CT, FISH, TP53 mutation analysis Exclude Richter’s Transformation Venetoclax + rituximab Yes [46]. Currently, results of a large clinical trial multicentre trial, with over 900 participants are awaited, comparing singleagent obinutuzumab and its combination with FC, chlorambucil or bendamustine in untreated and relapsed CLL is awaited; it is possible that obinutuzumab will replace rituximab in the long-standing FCR regime [47, 48]. Bruton Tyrosine Kinase Inhibitors—Ibrutinib The B cell receptor and downstream signalling pathways are crucial to the survival and proliferation of malignant cells in CLL. Unlike their healthy counterparts in CLL, signalling from the B cell receptor is activated independently of appropriate antigen stimulation—autologous activation. Bruton tyrosine kinase (BTK) which is only slightly downstream of the receptor itself activates the cell survival pathway NfK-B and MAP kinases, its inhibition leads to apoptosis of CLL cells [49]. Ibrutinib is an orally available small molecular inhibitor that binds to BTK preventing its kinase activity. This affects multiple signalling pathways and disrupts the interactions between CLL cells and the microenvironment leading to further apoptosis. Initially trialled in relapsed or refractory CLL, response rates to ibrutinib were between 84 and 97% and complete response rates were 12–23% [50]. This was independent of Has patient progressed on ibrutinib? No Ibrutinib Or Venetoclax + rituximab previously identified poor prognostic factors including advanced stage disease, number of previous lines of treatment and del 17(p). The RESONATE trial, a multicentre phase 3 open-label trial with 391 participants, compared ibrutinib with an anti-CD20 antibody, ofatumumab, note no longer in use, in a cohort of patients with relapsed CLL/SLL. Ibrutinib significantly improved the response rate, PFS and OS. The median PFS of 8.1 months in the ofatumumab group whilst at 9.4 months, the median PFS had not been reached in the ibrutinib group, PFS of 88% at 6 months [51]. RESONATE 2 showed that ibrutinib was more effective than single-agent chlorambucil as a first-line treatment in patients aged over 65. Ibrutinib significantly prolonged overall survival; estimated survival rate at 24 months was 98% with ibrutinib and 85% with chlorambucil, with a relative risk of death that was 84% lower in the ibrutinib group. The overall response rate was higher with ibrutinib than with chlorambucil (86% vs 35%) [51, 52]. It is important to note that unlike RESONATE in RESONATE 2, the presence of a 17p deletion was an exclusion criterion, and the use of single-agent chlorambucil without a CD20 antibody is now very rare. A more relevant comparison of ibrutinib versus bendamustine and rituximab in patients over 65 did show ibrutinib to confer a significant benefit. The estimated percentage of patients with progression-free survival at 2 years was 74% with bendamustine plus rituximab and 87% with ibrutinib alone. Curr Oncol Rep (2020) 22: 36 Page 5 of 9 36 Combined rituximab and ibrutinib provided no additional benefit compared with ibrutinib alone with an estimated PFS of 88% at 2 years [53]. Ibrutinib and obinutuzumab does appear to be beneficial compared with CLB-obinutuzumab with a high estimated 30-month overall survival and fewer serious adverse events in the iLLUMINATE phase 3 trial of first-line treatment [54] (Table 1). The use of ibrutinib first line in younger patients, without 17p deletion or p53 mutation, has not yet been clearly demonstrated, one trial has reported an improvement in PFS and OS with ibrutinib and rituximab compared with FCR as a firstline treatment of CLL in patients under 70 [55]. However, as of yet, there has only been a short follow-up period, and there was a surprisingly high number of deaths in the FCR arm indicating further work is needed. The results of the RESONATE trials have not been replicated in the clinic; the UK ibrutinib real world study reported that 44% of the patients had a dose reduction, interruption of complete cessation in the first 12 months compared with 4% in the resonate study. The OS at 12 months was 83%, 89% for patients with no dose reduction or cessation of less than 14 days compared with 90% in the RESONATE [56•]. A theoretical benefit of small molecule inhibitors is the reduced side effect profile but due to off-target effects, these are still not negligible with significant bleeding, recurrent infections and cardiac Table 1 toxicity, particular atrial fibrillation, being the most common reasons for treatment interruption or cessation. We are now beginning to understand the mechanisms underlying the ibrutinib failure to ibrutinib failure. Comparisons of targeted deep sequencing before initiation of ibrutinib and at the point or either CLL progression or Richter’s transformation identified new mutations in BTK or PLG2 that were not present prior to treatment [57]. A larger prospective study also identified these mutations in some patients who had not yet shown signs of clinical relapse suggesting sequencing may become an indicator of when further intervention is required [58]. Phosphatidylinositol 3-Kinases(PI3K) Inhibitors—Idelalisib The PI3K signalling pathway, downstream of the B cell receptor, is constitutively activated in CLL and is required for their survival and proliferation [59, 60]. Idelalisib is a potent and specific inhibitor of PI3K isoform expression of which is restricted to cells of haematopoietic origin. Idelalisib induces apoptosis in CLL cells whilst T cells and NK cell are unaffected. Like ibrutinib, idelalisib has multiple mechanisms of action, such as disruption of the CLL cell CXC12 and CXC13 driven chemotaxis towards stromal cells and their migration beneath them; this may keep the cells within the peripheral Significant randomised and more recent phase 1/2 trials using targeted small molecular inhibitors in CLL Treatment Randomised phase 3 trials Ibrutinib Ofatumumab Ibrutinib Chorambucil Ibrutinib Ibrutinib/rituximab Ibrutinib/obinutuzumab CLB/obinutuzumab Bendamustine/rituximab Ibrutinib/rituximab FCR Idelalisib/rituximab Rituximab Venetoclax/rituximab Bendamustine/rituximab Phase 1/2 trials Ibrutinib/venetoclax Alcalabrutinib FL/ RR Number Age ORR NMRD 2YOS Reference RR 195 196 136 133 67 67 73 72 63% 4% 86% 35% NR NR NR NR 1 year 90% 1 year 81% 98% 85% Byrd 2014 [51] 182 182 113 116 183 354 175 110 110 194 195 71 71 70 72 70 58 57 71 71 65 65 93% 94% 88% 73% 81% 96% 81% 81% 13% 92% 72% 1% 4% 35% 25% 8% 8% 59% NR NR 62% 13% 90% 94% 30 m 86% 30 m 85% 95% 3 years 99% 3 years 92% 1 year 92% 1 year 80% 92% 87% Woyach 2018 [53] 80 61 65 62 88% 95% 61% NR 1 year 99% 1 year 100% FL FL FL FL RR RR FL RR Burger 2015 [52] Moreno 2019 [54] Shanfelt 2019 [55] Furman 2014 [62] Seymour 2018 [68] Jain 2019 [73] Byrd 2016 [75] FL first-line therapy, RR relapsed or refractory, Age median in years, ORR overall response rate, NMRD negative MRD in peripheral blood, 2YOS 2-year overall survival rate unless alternate follow-up period specified 36 Page 6 of 9 blood increasing their susceptibility to apoptosis induction [60]. Idelalisib was initially evaluated in relapsed and refractory disease including patients with adverse features—bulky lymphadenopathy, 17p deletion/Tp53 mutation, IGHVunmutated and failure of multiple treatments. Idelalisib had an overall response rate of 72% in this cohort and PFS of 15.8 months [61]. The combination of idelalisib with rituximab, compared with rituximab alone, leads to higher overall response rate 81%vs 13% and a 12-month survival or 92% vs 81%. There was also a higher rate of reported serious adverse events in the idelalisib and rituximab group (40%)—the most common being pneumonia, pyrexia and febrile neutropoenia; it is likely that due to its toxicity, its use will be restricted to relapsed disease [62]. BCL2 Inhibition—Venetoclax An ability to evade apoptosis is required for the development of cancer—making its regulatory pathways an important therapeutic target [63]. Venetoclax, a BH3 mimic, prevents the interaction between BCL2 and BH3 inducing cell death [64]. Earlier BH3 mimetics showed good disease response but induced severe thrombocytopaenia in a phase 1 trial [65]. Venetoclax avoids this due to its higher specificity for BCL2 than some of its predecessors. The phase 1 and 2 trials of venetoclax showed impressive results with an overall response rate of 70–80% across all prognostic groups including patients with a 17p deletion/Tp53 mutation. Additionally, unlike the use of ibrutinib and idelalisib, venetoclax-induced MRD negative complete responses [66]. The most important adverse effect was the occurrence of fatal tumour lysis syndrome in the initial phase 1 trial; this could occur after even a single dose of 100 or 200 mg. Since the introduction of a strict dosing increment regime, there have been no further deaths attributable to, and a lower incidence of TLS. Monitoring for cytopaenias, infection and hepatotoxicity is also required. A retrospective analysis of the UK patients started on venetoclax, who had failed a BTK inhibitor and/or a PI3K inhibitor, reported an overall response rate of 88%. At the median follow-up of 15·6 months, the 1-year PFS and OS was 65.0% and 75.1%, respectively. Of particular interest was the response rate of 80% in patients who had received both BTK and PI3K inhibitors—a group of patients who previously had no further treatment options other than autologous stem cell transplant [67]. A similar study conducted in the US reported a lower ORR of 72%, but a much higher proportion of patients therapy was held/stopped 29% compared with 8% in the UK, and so far, the follow-up period has been significantly shorter—7 months [3]. The use of venetoclax in combination with monthly rituximab had a dramatically improved outcome at 2 years compared with BR in relapsed CLL, and this combination is now commonly used in clinical practice Curr Oncol Rep (2020) 22: 36 [68••]. Interestingly, the VR regimes result in 62% of patients having MRD negativity in peripheral blood, compared with 13% of the BR, hopefully longer follow-up will show that this combination results in a durable remission. Venetoclax is not immune to the development of resistance with identified mechanisms being mutation of BCL2 or the compensatory over expression of MCL1, another BCL family member [69, 70]. However with the promising MRD negativity seen in VR regimes, it is hoped that combination therapy that includes venetoclax may prevent the development of resistance and treatment failure. As the apoptotic pathways are targeted by venetoclax are universal, there is hope its success in CLL will be replicated. Within haematology, there have been promising results supporting the use of venetoclax in combination with hypomethylating agents such as daunorubicin in relapsed AML or with cytidine analogues as a first-line treatment for AML in older patients [71]. Conclusion—the Future of CLL Treatment The last decade has seen dramatic change in CLL treatment but there are important trial results awaited in 2020 and further into the future. The watch and wait principle of CLL— delaying treatment initiation until a progression means the iwCLL criteria are met is from the chemoimmunotherapy era. Several trials challenging this with the initiation of ibrutinib at diagnosis in patients without an indication to treat but high-risk disease are active, with results due to be reported in mid-2020 or in ongoing recruitment [72, 73]. Additionally, longer term follow-up in patients treated with small molecule inhibitors is required to see if the achievement of MRD negativity improves survival as seen with FCR—it is likely that monitoring MRD will become more common place in CLL, as it is already in other haematological malignancies [74]. The results of a multicentre phase 1/2 trials establishing the maximum tolerated dose of alcalabrutinib—a BTK inhibitor with higher specificity and lower reversibility than ibrutinib—are due to be published in early 2021 indicating that with time, we will likely see an increase in the number of available drugs within the classes of small molecule inhibitors already established [75]. Perhaps, the most exciting current trials in CLL are the use of multiple molecular inhibitors—such as venetoclax and ibrutinib simultaneous—this combination has been shown to provoke a complete remission, including MRD negativity, in patients after a limited duration of treatment meaning that patients may not be committed to indefinite therapy [73••]. Limiting length of treatment regimens has the advantageous of reduced exposure, and therefore, toxicity particularly relevant in CLL due to the increasingly complex comorbidities seen in this patient cohort. It is recommend that fit patients with relapsed CLL, or a 17p deletion, should be Curr Oncol Rep (2020) 22: 36 considered for allogeneic transplantation after the failure of one kinase inhibitor—whilst venetoclax provides a valid option for these patients in the future CAR T cells may also prevent the need for transplant [76]. Small studies of antiCD19 CAR T cells in patients who relapsed on BTK inhibitors have shown response rates of over 70% and a survival rate of 100%, although only after 6 months of follow-up. Further larger trials are required, but we can be cautiously optimistic; this may provide a safer alternative to transplantation in patients who fail small molecule therapy [77, 78]. Funding Information Dr. Milne is funded by a National institute for Health Research (NIHR) Academic Clinical Fellowship award. Dr. Chevassut is supported by a project grant from Bloodwise, UK. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. Page 7 of 9 36 4. 5. 6. 7. 8. 9. 10. 11. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance. Blood. 2009;114(20):4441–50. 30. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 1999;94(11):3658–67. 31. Strati P, Jain N, O’Brien S. Chronic lymphocytic leukemia: diagnosis and treatment. Mayo Clin Proc [Internet]. 2018;93(5):651–64. Available from: https://doi.org/10.1016/j.mayocp.2018.03.002. 32. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 2018;131(25):2745–60. 33. Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, Kay N, et al. Diagnostic criteria for monoclonal B-cell lymphocytosis. 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Chemotherapy combination treatment regimens with fludarabine in chronic lymphocytic leukemia. Hematol J. 2004;5(Suppl. 1). 41. Catovsky D, Richards S, Matutes E, Oscier D, Dyer M, Bezares R, et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 trial): a randomised controlled trial. Lancet. 2007;370(9583):230–9. 42. Rossi D, Terzi-Di-Bergamo L, De Paoli L, Cerri M, Ghilardi G, Chiarenza A, et al. Molecular prediction of durable remission after first-line fludarabinecyclophosphamide-rituximab in chronic lymphocytic leukemia. Blood. American Society of Hematology. 2015;126:1921–4. 43.• Fischer K, Bahlo J, Fink AM, Goede V, Herling CD, Cramer P, et al. Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL: updated results of the CLL8 trial. Blood. 2016:208–15 Update on long term outcomes of patients in the CLL8 trial showing that FCR results in lastining remission in patients without 17p deletion of P53 mutation and IGHV-mutated CLL as well as in patients who achieved MRD negativity in peripheral blood following 6 cycle of FCR. 44. Eichhorst B, Fink AM, Bahlo J, Busch R, Kovacs G, Maurer C, 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(7):928–42. 45. Michallet AS, Aktan M, Hiddemann W, Ilhan O, Johansson P, Laribi K, et al. Rituximab plus bendamustine or chlorambucil for chronic lymphocytic leukemia: primary analysis of the randomized, open-label mable study. Haematologica. 2018;103(4):698–706. 46. Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med [Internet]. 2014 Mar 20 [cited 2019 Nov 17];370(12):1101–10. Available from: http://www. nejm.org/doi/10.1056/NEJMoa1313984 47. Stilgenbauer S, Leblond V, Foà R, Böttcher S, Ilhan O, Knauf W, et al. Obinutuzumab plus bendamustine in previously untreated patients with CLL: a subgroup analysis of the GREEN study. Leukemia. 2018;32(8):1778–86. 48. A safety and efficacy study of obinutuzumab alone or in combination with chemotherapy in participants with chronic lymphocytic leukemia - Full Text View - ClinicalTrials.gov [Internet]. [cited 2019 Nov 17]. Available from: https://clinicaltrials.gov/ct2/show/ NCT01905943 49. Petlickovski A, Laurenti L, Li X, Marietti S, Chiusolo P, Sica S, et al. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood. 2005;105(12):4820–7. 50. Advani RH, Buggy JJ, Sharman JP, Smith SM, Boyd TE, Grant B, et al. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol. 2013;31(1):88–94. Curr Oncol Rep (2020) 22: 36 51. 52. 53. 54. 55. 56.• 57. 58. 59. 60. 61. 62. 63. 64. 65. Byrd JC, Brown JR, O’Brien S, Barrientos JC, Kay NE, Reddy NM, et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014;371(3):213–23. Burger JA, Tedeschi A, Barr PM, Robak T, Owen C, Ghia P, et al. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. N Engl J Med. 2015;373(25):2425–37. Woyach JA, Ruppert AS, Heerema NA, Zhao W, Booth AM, Ding W, et al. Ibrutinib regimens versus chemoimmunotherapy in older patients with untreated CLL. N Engl J Med [Internet]. 2018 Dec 27 [cited 2019 Nov 18];379(26):2517–28. Available from: http://www. nejm.org/doi/10.1056/NEJMoa1812836 Moreno C, Greil R, Demirkan F, Tedeschi A, Anz B, Larratt L, 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(1):43–56. Shanafelt TD, Wang XV, Kay NE, Hanson CA, O’Brien S, Barrientos J, et al. Ibrutinib–rituximab or chemoimmunotherapy for chronic lymphocytic leukemia. N Engl J Med. 2019;381(5): 432–43. Leukemia CL. Ibrutinib for relapsed/refractory chronic lymphocytic leukemia: a UK and Ireland analysis of outcomes in 315 patients. Haematologica. 2016;101(12):1563–72 Real world Ibrutinib outcomes in patients in the UK concluding that whilst effective it is much more likely for patients to have the drug held or stopped outwith trial settings. Woyach JA, Furman RR, Liu TM, Ozer HG, Zapatka M, Ruppert AS, et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286–94. Woyach JA, Ruppert AS, Guinn D, Lehman A, Blachly JS, Lozanski A, et al. BTKC481S-mediated resistance to ibrutinib in chronic lymphocytic leukemia. J Clin Oncol. 2017;35(13):1437– 43. Okkenhaug K, Vanhaesebroeck B. PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol. European Association for Cardio-Thoracic Surgery. 2003;3:317–30. Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, Keating MJ, et al. The phosphoinositide 3′-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood. 2011;118(13): 3603–12. Brown JR, Byrd JC, Coutre SE, Benson DM, Flinn IW, WagnerJohnston ND, et al. Idelalisib, an inhibitor of phosphatidylinositol 3-kinase p110δ, for relapsed/refractory chronic lymphocytic leukemia. Blood. 2014;123(22):3390–7. Furman RR, Sharman JP, Coutre SE, Cheson BD, Pagel JM, Hillmen P, et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med [Internet]. 2014 Mar 13 [cited 2019 Nov 17];370(11):997–1007. Available from: http://www. nejm.org/doi/10.1056/NEJMoa1315226 Hanahan D, Weinberg RA. Leading Edge Review Hallmarks of Cancer: The Next Generation; 2011. Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013;19(2):202–8. Roberts AW, Seymour JF, Brown JR, Wierda WG, Kipps TJ, Khaw SL, et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol. 2012;30(5):488–96. Page 9 of 9 36 66. Stilgenbauer S, Eichhorst B, Schetelig J, Coutre S, Seymour JF, Munir T, et al. Venetoclax in relapsed or refractory chronic lymphocytic leukaemia with 17p deletion: a multicentre, open-label, phase 2 study. Lancet Oncol. 2016;17(6):768–78. 67. Eyre TA, Kirkwood AA, Gohill S, Follows G, Walewska R, Walter H, et al. Efficacy of venetoclax monotherapy in patients with relapsed chronic lymphocytic leukaemia in the post-BCR inhibitor setting: a UK wide analysis. Br J Haematol. 2019;185(4):656–69. 68.•• Seymour JF, Kipps TJ, Eichhorst B, Hillmen P, D’Rozario J, Assouline S, et al. Venetoclax–rituximab in relapsed or refractory chronic lymphocytic leukemia. N Engl J Med. 2018;378(12):1107– 20 Phase 3 randomised trial in relapsed/refractory CLL that showed venetoclax and rituximab had a higher response rate, overall survial and propotion of patient wih undectable minimal residual disease compared with bendamustine and rituximab. 69. Blombery P, Anderson MA, Gong JN, Thijssen R, Birkinshaw RW, Thompson ER, 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(3): 342–53. 70. Bojarczuk K, Sasi BK, Gobessi S, Innocenti I, Pozzato G, Laurenti L, et al. BCR signaling inhibitors differ in their ability to overcome Mcl-1-mediated resistance of CLL B cells to ABT-199. Blood. 2016;127(25):3192–201. 71. DiNardo CD, Pratz K, Pullarkat V, Jonas BA, Arellano M, Becker PS, et al. Venetoclax combined with decitabine or azacitidine in treatment-naive, elderly patients with acute myeloid leukemia. Blood. 2019;133(1):7–17. 72. Ibrutinib as early therapy in chronic lymphocytic leukemia (CLL) Full Text View - ClinicalTrials.gov [Internet]. [cited 2019 Nov 17]. Available from: https://clinicaltrials.gov/ct2/show/NCT03207555 73.•• Jain N, Keating M, Thompson P, Ferrajoli A, Burger J, Borthakur G, et al. Ibrutinib and venetoclax for first-line treatment of CLL. N Engl J Med. 2019;380(22):2095–103 Phase 2 study showing combined ibrutinib and venetoclax in older untreated patients with high-risk CLL results in 62% having undetetable minimal residual disease. 74. Burger JA, Barr PM, Robak T, Owen C, Ghia P, Tedeschi A, et al. Long-term efficacy and safety of first-line ibrutinib treatment for patients with CLL/SLL: 5 years of follow-up from the phase 3 RESONATE-2 study. Leukemia [cited 2019 Nov 17]. https://doi. org/10.1038/s41375-019-0602-x. 75. Byrd JC, Harrington B, O’Brien S, Jones JA, Schuh A, Devereux S, et al. Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia. N Engl J Med. 2016;374(4):323–32. 76. Dreger P, Schetelig J, Andersen N, Corradini P, Van Gelder M, Gribben J, et al. Managing high-risk CLL during transition to a new treatment era: stem cell transplantation or novel agents? Blood. American Society of Hematology. 2014;124:3841–9. 77. Turtle CJ, Hay KA, Hanafi LA, Li D, Cherian S, Chen X, 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(26):3010–20. 78. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725–33. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. CENTENARY REVIEW ARTICLE Chronic lymphocytic leukemia: from molecular pathogenesis to novel therapeutic strategies Ferrata Storti Foundation Julio Delgado,1,2,3 Ferran Nadeu,2,3 Dolors Colomer,2,3,4 and Elias Campo2,3,4 1 Department of Hematology, Hospital Clínic, University of Barcelona, Barcelona; 2Centro de Investigación Biomédica en Red en Oncologia (CIBERONC), Madrid; 3Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona and 4 Hematopathology Section, Hospital Clínic, University of Barcelona, Barcelona, Spain ABSTRACT C hronic lymphocytic leukemia is a well-defined lymphoid neoplasm with very heterogeneous biological and clinical behavior. The last decade has been remarkably fruitful in novel findings, elucidating multiple aspects of the pathogenesis of the disease including mechanisms of genetic susceptibility, insights into the relevance of immunogenetic factors driving the disease, profiling of genomic alterations, epigenetic subtypes, global epigenomic tumor cell reprogramming, modulation of tumor cell and microenvironment interactions, and dynamics of clonal evolution from early steps in monoclonal B-cell lymphocytosis to progression and transformation into diffuse large B-cell lymphoma. All this knowledge has offered new perspectives that are being exploited therapeutically with novel, targeted agents and management strategies. In this review we provide an overview of these novel advances and highlight questions and perspectives that need further progress to translate this biological knowledge into the clinic and improve patients’ outcome. Haematologica 2020 Volume 105(9):2205-2217 History Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy characterized by the proliferation and accumulation of mature CD5+ B cells in the blood, bone marrow and lymphoid tissues. The diagnosis of CLL requires the presence of ≥5 x109/L monoclonal B cells of typical phenotype in the blood. Patients with C, glycine-arginine) of IGLV3-2101 mediated by somatic hypermutation confers autonomous BCR signaling.24 This change is present in 7-18% of CLL and seems responsible for the adverse outcome associated with the use of IGLV3-21 independently of the mutational status of the IGHV.24,25 Epigenetic studies have shown that, although both CLL subtypes are antigen-experienced, M-CLL keeps a methylation signature of germinal center-experienced cells (memory-like B cells), whereas U-CLL has a pre-germinal center, naïve-like methylation signature.5,26 Of note, these epigenetic studies also identified a third subtype with an intermediate profile made of cases with moderate IGHV mutation levels. All three epigenetic subsets have different usage of IGHV genes, stereotypes, genomic aberrations and clinical outcome (Table 1).27 Their prognostic relevance has been validated in retrospective cohorts and clinical trials.26–28 The intermediate epigenetic subtype may be more heterogeneous than initially thought since it includes most stereotype subset 2 cases with aggressive behavior whereas other cases may behave more indolently. The understanding of the biological significance of this subtype requires further analysis. The microenvironment in chronic lymphocytic leukemia CLL cells are highly dependent on signals coming from the microenvironment for proliferation and survival. Tumor cells proliferate primarily in lymph nodes, and to less extent in bone marrow,29 where they are in intimate contact with extracellular matrix, T cells, nurse-like cells, follicular dendritic cells and other stromal cells (Figure 2). The interactions between CLL cells and this complex microenvironment are mediated by a network of adhesion molecules, cell surface ligands, chemokines, cytokines, and their respective receptors. CLL cells organize their supportive inflammatory milieu and promote an immunosuppressive microenvironment through different mechanisms, such as secretion of soluble factors, cell-tocell contact, and release of extracellular vesicles (Figure 2).29,30 Environmental or self-antigens and homotypic interactions trigger BCR and Toll-like receptor (TLR) signaling, amplifying the response of CLL cells to other signals from the microenvironment and increasing the activation of anti-apoptotic and proliferation pathways.31,32 Genomic studies have identified recurrent mutations in genes regulating tumor cell-microenvironment interactions, which are already required for tumor cell growth. Thus, NOTCH1 mutations are dependent on the presence of Notch ligands in the microenvironment and activate processes such as cell migration, invasion and angiogenesis.33,34 BCR and NOTCH1 pathways are functionally linked, mutually enhancing their activation.35 MYD88 mutations activate the NF-κB pathway in response to TLR ligands, increasing the cytokine release involved in recruiting stromal and T cells.36 Tumor cells also reconfigure the function of T- and myeloid-derived cells towards a leukemia-supportive and immunosuppressive microenvironment.30,37 Thus, tumor cells reduce T-cell motility and the effector function of CD4+ cells while inducing CD8+-cell exhaustion38–41 and monocyte differentiation towards macrophages with protumoral functions (M2like) and nurse-like cells.37 Many studies have confirmed the fundamental role of BCR activation for CLL pathogenesis.42 Several proteins, haematologica | 2020; 105(9) including phosphatidylinositol 3 kinase (PI3K), Bruton tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) are essential for BCR signal transduction.43 The effect of BCR-mediated signaling varies according to IGHV mutation status: M-CLL cells are generally driven towards anergy, whereas U-CLL cells are more directed towards cell growth and proliferation.44 Moreover, anergic cells normally retain a higher susceptibility to apoptosis unless anti-apoptotic proteins such as BCL2 are overexpressed, as is the case for CLL cells.45 Indeed, most major therapeutic advances occurring in the last decade are related to the inhibition of BCR and BCL2-mediated signaling. Structural genomic aberrations Initial chromosome banding analysis revealed that deletions or trisomies were relatively common but only observed in fewer than half of the patients.46 With the advent of fluorescent in situ hybridization (FISH), genomic aberrations were identified in more than 80% of patients, the more relevant being trisomy 12, 13q deletion [del(13q)], 11q deletion [del(11q)] and 17p deletion [del(17p)];47 and FISH became the gold standard for genomic evaluation in CLL. Later, the introduction of more effective mitogens expanded the use of chromosome banding analysis in CLL and revealed other aberrations that could not be detected by FISH, including chromosome translocations in 20-35% of the cases.48 These translocations may occur in the context of complex karyotypes. The most common rearrangements involve 13q14, with multiple partners, and the IGH locus. The genes most commonly rearranged with IGH are BCL2 [t(14;18)(q32;q21)] (2% of cases, usually M-CLL);3,49 and BCL3 [t(14;19)(q32;q13)] or BCL11A [t(2;14)(p16;q32)] (5%) in newly diagnosed patients are highlighted in bold. POT1 in U-CLL), whereas others are seen in both subtypes; (ii) age of the patients: MYD88 mutations seem to be more frequent in younger patients; (iii) disease evolution: some mutations (SF3B1, POT1, ATM) are more frequent in patients requiring therapy compared to those with stable disease, and some others (TP53, BIRC3, MAP2K1, NOTCH1) are more frequent in patients with progressive disease after chemoimmunotherapy (CIT).3,4,59 The co-occurrence of many of these driver alterations within the same tumor complicates the analysis of their relative clinical relevance. For instance, mutations in SF3B1, POT1 or XPO1 are generally associated with poor prognosis, but they rarely appear on their own.13 This has led some investigators to propose a multi-hit model in which the accumulation of driver mutations, regardless of the individual genes targeted by each of these mutations, gradually impairs patients’ outcome.3,13 Indeed, the survival of patients in whom no driver aberrations are identified is comparable to that of individuals in the general population, further reinforcing this concept.3 Deep, targeted next-generation sequencing has revealed that subclonal mutations (i.e., those present in only a fraction of tumor cells) can be detected for all driver genes and are associated with rapid disease progression haematologica | 2020; 105(9) and poor outcome.11–13 This is particularly relevant for TP53 mutations given the fact that, as explained below, CLL therapy is based on the presence or absence of these mutations. The current consensus is that, apart from clonal mutations, subclonal mutations with a variant allelic frequency ranging from 5 to 10% (and therefore below the threshold of detection by conventional molecular techniques) could also be reported, whereas those with a variant allelic frequency lower than 5% should not, but there is much controversy around these issues and this recommendation may well change in the future.60,61 Furthermore, the analysis of clonal and subclonal aberrations has also allowed the reconstruction of each tumor’s phylogeny. Thus, clonal aberrations, which are mostly structural abnormalities [e.g. trisomy 12, del(13q)] generally correspond to earlier driving events, while subclonal mutations in driver genes (e.g., SF3B1, POT1, NOTCH1) are acquired later over the course of the disease.13 Moreover, some genes appear to be specifically selected at relapse. For instance, small clones harboring TP53 mutations typically expand and dominate the disease after CIT, which explains the poor prognosis associated with these subclonal mutations.12,62 Apart from TP53, mutations in IKZF3 and SAMHD1 have also been recur2209 J. Delgado et al. rently selected in small cohorts of patients after CIT.63,64 Clonal evolution plays an important role not only in resistance to CIT, but also to novel agents. Indeed, different point mutations have been identified in the BTK and PLCG2 genes in patients previously treated with the BTK inhibitor ibrutinib,65 and in the BCL2 gene in patients relapsing after treatment with the BCL2 antagonist venetoclax.66 Resistance to these agents has been associated with these mutations in around 70% of cases, although they are usually subclonal and their specific role causing resistance needs to be proven.67 Other resistance mechanisms involve upregulation of BCL-XL and MEK/ERK, and cell reprogramming and transdifferentiation to cell subtypes that do not require BCR signaling.65,67–70 Epigenomic landscape The genome of CLL features widespread hypomethylation, and a large fraction of the differences between UCLL and M-CLL can be attributed to their different cell of origin in germinal center-independent or -experienced B cells, respectively.5 Major hypomethylation changes occur at transcription factor binding sites such as TCF3, PU.1/SPIB, NFAT and EGR, and enhancers that modulate genes relevant for CLL pathogenesis involved in B-cell function, BCR signaling, and NF-κB activation among others. This methylation profile is already acquired at the MBL stage3 and remains relatively stable over time. However, some CLL have intratumor variability in certain regions, which may alter the expression of several genes and facilitate tumor evolution.71 Of note, this variability is greater in U-CLL than in M-CLL and is associated with increasing number of subclones.7,71 Several groups have evaluated the full reference epigenome of CLL, providing a genome-wide map of histone marks and three-dimensional chromatin architecture.6,72–74 Surprisingly, there was a significant variability in active regulatory regions among individual patients. This variability, and also the total number of active sites, was larger in U-CLL than in M-CLL. Around 80% of these active sites were also present in normal naïve, germinal center, memory, or plasma cells.6 Some of these active regions are seen in all CLL cases but in none of the normal B-cell subtypes, and may therefore be crucial for CLL pathogenesis. Most of these de novo active regions target regulatory loci and super-enhancers enriched in transcription binding motifs of NFAT, FOX, TCL/LEF, and PAX5, which have been shown to play a role in CLL pathogenesis and could potentially be targeted pharmacologically.26,72 Somatic mutations in chromatin remodeler genes could modify the epigenomic landscape of CLL, but they are uncommon in this malignancy compared to other lymphoid neoplasms. CHD2 is mutated in 5% of CLL and 7% of MBL.75 The histone methyltransferase SETD2 and ARID1A are also mutated in a small proportion of patients. Of note, MYD88 mutations and trisomy 12 are associated with specific remodeling of chromatin activation and accessibility regions. More specifically, the epigenomic profile induced by MYD88 mutations targets regulatory regions related to NF-κB signaling,6 whereas the epigenetic configuration of trisomy 12 CLL is characterized by a subtype-specific hypomethylation signature associated with increased H3K27 acetylation, which leads to the overexpression of 25 target genes including RUNX3.76 2210 Pathogenic mechanisms in the evolution of the disease CLL is always preceded by an often unnoticed premalignant state known as high-count MBL.77 Low-count MBL may persist for a long time but the risk of progression is negligible.78 Yearly, 1% of cases of high-count MBL evolve into CLL requiring therapy,79 and 2-10% of patients with symptomatic CLL eventually develop Richter transformation.80 At the other end of the spectrum, around 30% of patients with CLL never require any CLL-specific therapy and die of other causes, and 1-2% of them even experience spontaneous regression of their disease.81 It is therefore evident that the rate and pattern of growth (or even decline) of the disease can vary greatly among patients. Patients with high-count MBL carry mutations in driver genes which may be detected at a median of 41 months prior to progression to CLL.3,82 The mutation rates for the most common drivers (e.g., SF3B1, DDX3X, BIRC3, ATM) are comparable between MBL and CLL, with only a few genes being more commonly mutated in CLL (NOTCH1, TP53, XPO1).82,83 Patients with MBL with mutated drivers have a shorter time to first treatment compared to cases without mutations. Once CLL is established, the growth dynamics of tumor cells is heterogeneous. Some patients exhibit a logistic-like behavior in which the clone stabilizes over time, whereas some others show an exponential-like growth pattern.84 This exponential growth, clinically defined as “short lymphocyte doubling time” is still considered an adverse prognostic parameter in CLL.85,86 As expected, the median number of driver mutations (both clonal and subclonal) is higher in patients with exponential growth, and this patient population also displays unmutated IGHV genes more frequently. In addition, the rate of clonal evolution after therapy (i.e., with a significant shift in at least one subclone) is also higher in patients with exponential growth (Figure 4). Transformation of CLL into an aggressive lymphoma occurs in 2-10% of patients and in most of them (>90%) corresponds to a DLBCL, but Hodgkin lymphoma may also occur.87 The DLBCL usually emerges as a linear evolution of the same CLL clone with only rare cases deriving from a branching divergent subclone.88 CLL carrying stereotyped subset 8 (IGHV4-39), NOTCH1 or TP53 mutations and complex karyotypes are at higher risk of transformation after CIT. Transformed DLBCL frequently add CDKN2A deletions and MYC translocations or amplifications on top of the genomic alterations already present in the original CLL, but lack the common mutations observed in primary DLBCL indicating that they may correspond to a different biological category.80 Richter transformation also occurs in patients treated with BTK inhibitors. These tumors do not usually acquire BTK or PLCG2 mutations but, if these were present in the original CLL, subclones may emerge with additional independent mutations.89,90 In the rare instances in which the disease regresses spontaneously, the patients uniformly have mutated IGHV genes, no stereotypes, low proliferative activity and poor migration to proliferation centers as exemplified by a high CXCR4 expression. Interestingly, patients with spontaneous regression show reduced T-cell exhaustion and increased T-cell proliferation, confirming the important role of the immune system in CLL progression.91 Moreover, driver mutations are also present in these patients but they always remain stable without subclonal shifts.91 haematologica | 2020; 105(9) CLL pathogenesis and management Figure 4. Evolutionary steps and growth dynamics of chronic lymphocytic leukemia. (Left) The progression of monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) is a linear process discriminated by the total number of lymphocytes. The presence of driver alterations is associated with rapid progression. Although a few alterations are enriched in CLL compared to MBL, both phases share a similar driver composition. (Middle) The main growth dynamics during the pre-treatment phase of the CLL are shown, including spontaneous regression, logistic growth and exponential growth. The main characteristics associated with each pattern are specified. WBC; peripheral white blood cell count. (Right) Richter transformation to diffuse large B-cell lymphoma (DLBCL) is associated with subset #8, NOTCH1 or TP53 mutations and complex karyotype. It follows a linear evolution from the CLL clone through the recurrent acquisition of CDKN2A and MYC alterations. Clinical and prognostic implications of novel discoveries The clinical course of CLL is rather heterogeneous, ranging from a fairly asymptomatic disease that may even regress spontaneously to a progressive disease that eventually leads to the patient’s death, so there has always been remarkable interest in determining the prognosis of individual patients. Even though many prognostic markers have been identified over the past decades, only a few prevail. For many years, the prognosis of patients with CLL was defined using purely clinical parameters, such as those included in the Rai and Binet staging systems,92,93 the IGHV mutational status,17,18 and numerical aberrations as determined by FISH.47 With the advent of next-generation sequencing, novel drivers were discovered (NOTCH1, SF3B1, BIRC3) and incorporated into these prognostic systems, but none of these attempts succeeded in becoming standard of care.94–96 Indeed, the International Workshop on CLL (iwCLL) guidelines only recommend evaluating the IGHV status and presence/absence of TP53 aberrations in routine practice.86 The recent CLL International Prognostic Index (CLL-IPI) incorporates both clinical and cytogenetic/genomic data (age, clinical staging, β2microglobulin serum concentration, IGHV mutation status and TP53 aberrations) into one prognostic score.97 The CLL-IPI was developed in cohorts of patients treated with CIT and has been validated in retrospective series.98–100 Among the five items, both TP53 and IGHV have the strongest impact on a patient’s outcome, and it is therefore not surprising that simplified versions of the CLL-IPI incorporating only these two markers have been proposed.101 A recent study has determined that a score based on the presence of unmutated IGHV, absolute lymphocyte count >15 x109/L, and palpable lymph nodes predicts for a shorter time to first treatment in patients with early, asymptomatic disease.102 On the other hand, several groups are advocating for the incorporation of novel markers, such as a complex karyotype55 or epigenetic subsets,27,28 into clinical practice. All these novel prognostic haematologica | 2020; 105(9) and/or predictive models will need to be validated in cohorts of patients treated with targeted agents. Treatment Treatment for CLL has changed remarkably in the last decade (Figure 5). The mainstay of therapy used to be CIT - a combination of conventional chemotherapeutic agents plus a monoclonal antibody, such as rituximab or obinutuzumab, - although this is no longer the case, at least for most patients. Novel, targeted agents are now the preferred option, and among them, the drugs currently approved by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) are the BTK inhibitor ibrutinib, the BCL2 inhibitor venetoclax and the PI3K inhibitor idelalisib, while the second-generation BTK inhibitor acalabrutinib and PI3K inhibitor duvelisib have already been approved by the FDA and are under evaluation by the EMA. Frontline therapy Not all patients with CLL require therapy. Despite all recent advances, the iwCLL still recommends watchful observation for patients with asymptomatic disease.86 This recommendation is based on at least two randomized trials comparing observation to either chlorambucil monotherapy or fludarabine, cyclophosphamide and rituximab (FCR).103,104 Both trials concluded that early therapy in asymptomatic patients was not associated with a prolonged overall survival. Very recently, preliminary results from a third trial comparing ibrutinib versus observation were presented.105 Patients receiving ibrutinib had a longer event-free survival, but no overall survival advantage, although the results were still immature. Moreover, although severe adverse events rates were comparable between groups, patients receiving ibrutinib had a higher incidence of some specific adverse events such as bleeding, hypertension and atrial fibrillation. For patients with symptomatic disease requiring therapy, ibrutinib is often recommended based on four phase III 2211 J. Delgado et al. Figure 5. Recommended therapy for patients with symptomatic chronic lymphocytic leukemia. M-CLL, mutated IGHV; U-CLL, unmutated IGHV; FCR: fludarabine + cyclophosphamide + rituximab; BR: bendamustine + rituximab; V: venetoclax; VR: venetoclax + rituximab; VO: venetoclax + obinutuzumab; I: ibrutinib; IO: ibrutinib + obinutuzumab; A: acalabrutinib; ClbO: chlorambucil + obinutuzumab; R: rituximab; D: duvelisib; AlloHCT: allogeneic hematopoietic cell transplantation; R-CHOP, rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; IGHV, immunoglobulin heavy-chain variable region. Acalabrutinib (A) is approved for both treatment-naïve and relapsed disease by the FDA but not the EMA. #Venetoclax (V) monotherapy is approved for patients with TP53 aberrations who are refractory or intolerant to ibrutinib or patients without TP53 aberrations who are refractory or intolerant to both chemoimmunotherapy and ibrutinib. Venetoclax plus rituximab (VR) is approved for any patient with relapsed disease. †Duvelisib (D) monotherapy is approved for relapsed disease (minimum of two prior therapies) by the FDA but not the EMA. ‡AlloHCT is recommended for appropriate patients with high-risk disease, defined by TP53 aberrations and/or complex karyotype in whom ibrutinib and/or venetoclax has failed. Allogeneic HCT is also recommended for appropriate patients with transformed disease who have responded to salvage chemotherapy (e.g., R-CHOP). randomized clinical trials comparing ibrutinib with chlorambucil monotherapy106 and other commonly used CIT combinations, namely FCR, bendamustine plus rituximab and chlorambucil plus obinutuzumab (ClbO).107–109 Ibrutinib was superior to chlorambucil and all CIT combinations in terms of response rate and progression-free survival, and even conferred a longer overall survival compared to that provided by chlorambucil monotherapy and FCR.106,107 In these trials, ibrutinib was sometimes combined with a monoclonal antibody, either rituximab or obinutuzumab, and sometimes given as monotherapy, but the true added value of the monoclonal antibody in this context is unknown.108,110 In terms of toxicity, ibrutinib was less toxic than CIT combinations when severe adverse events or toxic deaths were considered.107–109 Apart from ibrutinib, patients with M-CLL, devoid of 2212 TP53 aberrations and fit enough to tolerate FCR therapy, may still be good candidates for the latter, with the benefit being that this treatment can be completed in 6 months while ibrutinib must be taken indefinitely. This option would be particularly valuable for non-compliant patients or those in whom ibrutinib is contraindicated. If FCR is the treatment of choice, caution must be taken in patients with NOTCH1 mutations, in whom rituximab appears to have little added value.59 Other genomic subgroups, such as patients with BIRC3 mutations appear to derive little benefit from CIT,111,112 but these results should be further validated. Unfit patients also have the alternative of venetoclax plus obinutuzumab (VO) as frontline therapy. This is based on a phase III trial that compared VO with ClbO in elderly/unfit patients.113 VO was superior in terms of haematologica | 2020; 105(9) CLL pathogenesis and management response rate and progression-free survival, and had a comparable safety profile. In this trial VO was administered for a definite period of time (2 years), which is quite appealing for older/unfit patients. Moreover, many well established adverse prognostic markers, including U-CLL, ATM aberrations or NOTCH1/BIRC3 mutations, lost their negative effect in patients treated with VO. The only factor that remained predictive of a shorter progression-free survival in this cohort of patients was TP53 aberrations.112 Finally, the alternative BTK inhibitor acalabrutinib was recently approved by the FDA (not by the EMA yet) as frontline therapy in view of the results of a phase III trial comparing acalabrutinib versus ClbO.114 Relapsed/refractory disease Treatment for relapsed/refractory disease must be decided depending on prior therapy and also the reason why the original treatment was no longer appropriate (e.g., refractoriness vs. intolerance). Ibrutinib is the current gold standard therapy for patients with relapsed/refractory disease, based on the results of several phase I-III trials,115–119 but this is also changing for two main reasons: (i) an increasing proportion of patients currently receive ibrutinib as frontline therapy; and (ii) a few serious contenders have appeared in the last year. Venetoclax is one of the best alternatives in this situation, including patients with high-risk genomic aberrations. The drug was already proven effective and safe in several phase I-II trials, in patients who had previously received either CIT or BTK/PI3K inhibitors.120–123 The formal confirmation of this promising activity came with a phase III trial in which venetoclax combined with rituximab was superior to bendamustine plus rituximab in terms of response rate, progression-free survival and overall survival, leading to its full approval for patients with relapsed/refractory CLL.124 Other possibilities are PI3K inhibitors and alternative BTK inhibitors. Idelalisib, in combination with rituximab, was the first PI3K inhibitor approved for the treatment of relapsed/refractory CLL based on the results of a phase III trial,125,126 and yet it is infrequently used because of its less favorable adverseevent profile. It may have a role in patients with complex karyotypes,127 who have a higher risk of progression and/or transformation when treated with ibrutinib or venetoclax,90,128 or in older patients who also tend not to tolerate ibrutinib well,129 but there are no randomized data to substantiate this potential superiority. Duvelisib was the second PI3K inhibitor approved by the FDA, also based on a phase III randomized trial.130 The efficacy and safety profile of the drug appear comparable with those of idelalisib, if not slightly advantageous. Regarding alternative BTK inhibitors, there are several products in development, but only acalabrutinib is approved by the FDA for the treatment of relapsed/refractory CLL. This is based on a phase III trial in which acalabrutinib was superior to either bendamustine plus rituximab or idelalisib plus rituximab.131 In this trial, prior ibrutinib therapy was not allowed, but a separate trial has shown that 85% of patients who were intolerant to ibrutinib were subsequently able to take acalabrutinib, with a 76% response rate.132 Despite all recent therapeutic advances, a proportion of patients will still fail to respond and should be considered for curative therapy. Currently, only allogeneic hematopoietic cell transplantation can be considered potentially curative, but it is also associated with considhaematologica | 2020; 105(9) erable morbidity and mortality. Over the past decades, the number of patients referred for allogeneic hematopoietic cell transplantation has dropped significantly,133 but the procedure should be recommended to young/fit patients in whom BCR/BCL2 inhibitor treatment fails, particularly in those with TP53 aberrations, or in the case of Richter transformation.134,135 Moreover, although chimeric antigen receptor T cells could also be appropriate in this situation and the results are promising,136 none of the commercially available products is yet approved for this indication. Disease transformation Richter transformation remains an ominous event for patients with CLL, particularly when it is clonally related to the original CLL, because none of the recently approved novel agents is truly effective. Indeed, disease transformation is a relatively common cause of failure to benefit from these drugs.90,128,129 Histological confirmation is always recommended since it can guide prognosis (i.e., Hodgkin lymphoma and clonally unrelated tumors have more favorable prognosis). Patients with transformed disease should be offered conventional CIT (e.g., RCHOP: rituximab plus cyclophosphamide, doxorubicin, vincristine, prednisone) followed by allogeneic hematopoietic cell transplantation in the case of response. Autologous hematopoietic cell transplantation remains an option if allogeneic transplantation is considered inappropriate.134 Chimeric antigen receptor T cells may also be effective but, unfortunately, none of the approved products is current available for patients with Richter transformation. Conclusions and perspectives Recent molecular studies have provided many insights into the processes that govern the development and progression of CLL, including many novel mutated genes clustered in different functional pathways. The CLL epigenome is reprogrammed through the modulation of regulatory regions that appear de novo in the disease, whereas other regions maintain functions already present in different stages of B-cell differentiation. Analysis of the CLL microenvironment has provided clues to understand the survival of tumor cells and resistance to therapy. All this knowledge has offered new perspectives that are being exploited therapeutically with novel agents and strategies. However, these studies are also raising new questions. The relationship between the remarkable molecular heterogeneity of the disease and the clinical diversity is not well understood. The disease is always preceded by a premalignant state (MBL) which shares most molecular drivers with overt CLL. In many cases, these molecular drivers remain constant over time. However, clonal evolution is also possible and is usually associated with exponential tumor growth, progressive disease and, in some cases, disease transformation. Most studies have been performed in pretreated patients and it is not fully understood how the genome and epigenomic alterations and microenvironmental interactions influence the evolution of the disease. Translating new knowledge into clinical practice will require an effort to obtain an integrated view of all these factors in order to understand the disease better and design effective treatments and management strategies. 2213 J. Delgado et al. Acknowledgments The authors would like to thank Silvia Beà, Jose Ignacio Martín-Subero, and Armando Lopez-Guillermo (Hospital Clinic of Barcelona and IDIBAPS) for their helpful comments on the manuscript, and Neus Giménez (IDIBAPS) for her assistance with the figure design. JD is supported by a grant from Generalitat de Catalunya (PERIS IPFE SLT006/17/301). FN is supported by a predoctoral fellowship from the Ministerio de Ciencia e Innovación (MCI) (BES2016-076372). DC is supported by MCI (RTI2018–094584B-I00), Generalitat de Catalunya Suport Grups de Recerca (AGAUR, grant 2017-SGR-1009) and CIBERONC (CB16/12/00334). EC is supported by grants from “La References 1. Campo E, Ghia P, Montserrat E, MüllerHermelink HK, Stein H, Swerdlow SH. Chronic lymphocytic leukaemia/small lymphocytic lymphoma. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Rev 4th Ed. Lyon: WHO Press; 2017:216-221. 2. Howlader N, Noone A, Krapcho M, et al. SEER Cancer Statistics Review, 1975-2016, National Cancer Institute. Bethesda, MD, USA. 3. Puente XS, Beà S, Valdés-Mas R, et al. Noncoding recurrent mutations in chronic lymphocytic leukaemia. Nature. 2015;526 (7574):519-524. 4. Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015;526(7574):525-530. 5. Kulis M, Heath S, Bibikova M, et al. Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic lymphocytic leukemia. Nat Genet. 2012;44(11):1236-1242. 6. Beekman R, Chapaprieta V, Russiñol N, et al. The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia. Nat Med. 2018;24(6):868-880. 7. Oakes CC, Claus R, Gu L, et al. Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia. Cancer Discov. 2014;4(3):348-361. 8. Berndt SI, Camp NJ, Skibola CF, et al. Metaanalysis of genome-wide association studies discovers multiple loci for chronic lymphocytic leukemia. Nat Commun. 2016;7(1): 10933. 9. Speedy HE, Beekman R, Chapaprieta V, et al. Insight into genetic predisposition to chronic lymphocytic leukemia from integrative epigenomics. Nat Commun. 2019;10(1): 3615. 10. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell. 2011;20(2):246-259. 11. Landau DA, Carter SL, Stojanov P, et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013;152(4):714-726. 12. Nadeu F, Delgado J, Royo C, et al. Clinical impact of clonal and subclonal TP53, SF3B1, BIRC3, NOTCH1, and ATM mutations in chronic lymphocytic leukemia. Blood. 2214 Caixa” Foundation (CLLEvolution-LCF/PR/HR17/ 52150017), the Instituto de Salud Carlos III and the European Regional Development Fund (FEDER – “Una Manera de Hacer Europa”) (PMP15/00007), MCI (grants RTI2018094274-B-I00 and SAF2016-81860-REDT), CIBERONC (CB16/12/00225) and AGAUR (2017-SGR-1142). EC is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats (ICREA) of the Generalitat de Catalunya.

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