Minimal residual disease in chronic lymphocytic ...

Critical Reviews in Clinical Laboratory Sciences

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Minimal residual disease in chronic lymphocytic leukemia: A consensus paper that presents the clinical impact of the presently available laboratory approaches Ciprian Tomuleasa, Cristina Selicean, Sonia Cismas, Anca Jurj, Mirela Marian, Delia Dima, Sergiu Pasca, Bobe Petrushev, Vlad Moisoiu, Wilhelm-Thomas Micu, Anna Vischer, Kanza Arifeen, Sonia Selicean, Mihnea Zdrenghea, Horia Bumbea, Alina Tanase, Ravnit Grewal, Laura Pop, Carmen Aanei & Ioana Berindan-Neagoe To cite this article: Ciprian Tomuleasa, Cristina Selicean, Sonia Cismas, Anca Jurj, Mirela Marian, Delia Dima, Sergiu Pasca, Bobe Petrushev, Vlad Moisoiu, Wilhelm-Thomas Micu, Anna Vischer, Kanza Arifeen, Sonia Selicean, Mihnea Zdrenghea, Horia Bumbea, Alina Tanase, Ravnit Grewal, Laura Pop, Carmen Aanei & Ioana Berindan-Neagoe (2018) Minimal residual disease in chronic lymphocytic leukemia: A consensus paper that presents the clinical impact of the presently available laboratory approaches, Critical Reviews in Clinical Laboratory Sciences, 55:5, 329-345, DOI: 10.1080/10408363.2018.1463508 To link to this article: https://doi.org/10.1080/10408363.2018.1463508

Published online: 25 May 2018.

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CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 2018, VOL. 55, NO. 5, 329–345 https://doi.org/10.1080/10408363.2018.1463508

REVIEW ARTICLE

Minimal residual disease in chronic lymphocytic leukemia: A consensus paper that presents the clinical impact of the presently available laboratory approaches Ciprian Tomuleasaa,b, Cristina Seliceana, Sonia Cismasc,d, Anca Jurje, Mirela Mariana, Delia Dimaa, Sergiu Pascae, Bobe Petrusheve, Vlad Moisoiue, Wilhelm-Thomas Micue, Anna Vischerd, Kanza Arifeend, Sonia Seliceand, Mihnea Zdrengheaa,d, Horia Bumbeaf,g, Alina Tanaseh, Ravnit Grewali, Laura Pope, Carmen Aaneij and Ioana Berindan-Neagoee a Department of Hematology, Ion Chiricuta Clinical Cancer Center, Cluj Napoca, Romania; bResearch Center for Functional Genomics and Translational Medicine/Hematology, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Romania; cDepartment of Genetics, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania; dDepartment of Hematology, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Romania; eResearch Center for Functional Genomics and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Romania; fDepartment of Hematology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; gDepartment of Hematology, University Clinical Hospital, Bucharest, Romania; h Department of Stem Cell Transplantation, Fundeni Clinical Institute, Bucharest, Romania; iSouth African Medical Research Council Bioinformatics Unit, The South African National Bioinformatics Institute (SANBI), University of the Western Cape, Bellville, South Africa; j Hematology Laboratory, Pole de Biologie-Pathologie, University Hospital of St. Etienne, St. Etienne, France

ABSTRACT

ARTICLE HISTORY

Chronic lymphocytic leukemia (CLL) is a malignancy defined by the accumulation of mature lymphocytes in the lymphoid tissues, bone marrow, and blood. Therapy for CLL is guided according to the Rai and Binet staging systems. Nevertheless, state-of-the-art protocols in disease monitoring, diagnostics, and prognostics for CLL are based on the assessment of minimal residual disease (MRD). MRD is internationally considered to be the level of disease that can be detected by sensitive techniques and represents incomplete treatment and a probability of disease relapse. MRD detection has been continuously improved by the quick development of both flow cytometry and molecular biology technology, as well as by next-generation sequencing. Considering that MRD detection is moving more and more from research to clinical practice, where it can be an independent prognostic marker, in this paper, we present the methodologies by which MRD is evaluated, from translational research to clinical practice.

Received 19 February 2018 Revised 18 March 2018 Accepted 8 April 2018 Published online 24 May 2018

Background on CLL and MRD Chronic lymphocytic leukemia (CLL) is a malignancy defined by the accumulation of mature lymphocytes in the lymphoid tissues, bone marrow and blood. CLL was first described in 1827 in patients presenting with enlarged lymph nodes and spleen and mature-looking lymphocytes in the peripheral blood and lymph nodes [1,2]. Later studies described the malignant cells as being of the B-lineage origin, with a special emphasis on surface immunoglobulins [3,4]. Arguments were raised regarding the monoclonal expansion of the mentioned cells. Malignant CLL cells express only one type of immunoglobulin chain idiotype and thus the tumor population undergoes clonal expansion [5–7]. The clinical staging for CLL was established in 1975 by Rai et al. considering lymphocytosis, enlarged lymph nodes, CONTACT Ciprian Tomuleasa

ciprian.tomuleasa@umfcluj.ro

ß 2018 Informa UK Limited, trading as Taylor & Francis Group

KEYWORDS

Chronic lymphocytic leukemia; minimal residual disease; clinical relevance

anemia and thrombocytopenia [8]. In the United States (US), CLL has an incidence of 4.1/100,000 people, with over 15,000 new cases and approximately 4500 diseaserelated deaths [9–11]. CLL incidence has a male to female ratio of 2:1 [12,13] and although hormones were not linked with disease incidence or survival, a higher survival rate has been reported in women, especially those with higher parity [14,15]. The highest level of correlation regarding CLL is with hereditary factors, as CLL is a frequent disease in the Western world but has a low incidence in Asian countries. For example, the frequency of CLL in Japan is 4–5 times lower than in the US [16,17]. A condition known to precede the development of CLL is monoclonal B-cell lymphocytosis [18,19]. Monoclonal B-cell lymphocytosis is rather frequent in individuals over 60 years of age and in families that

Department of Hematology, Ion Chiricuta Clinical Cancer Center, Cluj Napoca, Romania

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show a hereditary model for CLL [20–23]. Current National Comprehensive Cancer Network (NCCN) guidelines recommend classification from the point of view of prognosis in three groups: low, intermediate, and high risk [24]. Diagnosis of CLL is mainly based on laboratory findings – lymphocytosis, typically with small lymphocytes with condensed chromatin and light blue cytoplasm. Additional microscopy techniques can be used for research purposes, as is the case of immunofluorescence or electron microscopy [25–27]. Moderately condensed nuclei and prominent nucleoli are the hallmarks of atypical CLL or of the evolution towards Richter syndrome [28–30]. Bone marrow examination reveals degree and pattern of infiltration with leukemic cells. The diffuse pattern of bone marrow infiltration presents with a more aggressive form of the disease. Lymph node examination shows infiltration of leukemic cells, or, very rarely, shows cells that mimic phenotypically the Reed–Sternberg cells [31,32]. The next step in the diagnostic procedure is immunophenotyping. Typically, CLL leukemic cells express CD19 (B-cell lineage marker), CD5 (T-cell lineage marker), CD23, CD22, and CD79b. They have low expression of CD20 and surface immunoglobulins. Cells are negative for CD10 and CD103, the latter presenting expression just in hairy cell leukemia [33–37] and some cases of splenic diffuse red pulp small B-cell lymphoma. CLL cells are CD200 þ low, which is the most important marker to differentiate CLL from mantle cell lymphoma [38–42], while other groups also recommend the use of CD81, CD43 þ high, and receptor tyrosin kinase-like orphan receptor 1 (ROR1) as diagnostic markers [43,44]. The final step in CLL diagnosis consists of cytogenetic and molecular genetic analysis of the malignant clone. Some of the more frequent chromosome abnormalities are del (13q), trisomy 12, 11q-, del 6q, and 17p-. Deletions of 13q, especially 13q14 are found in most patients with CLL. Several genes are located in this region and are thus affected by the deletion. Some of these important genes include DLEU1, DLEU2, DLEU6, DLEU7, DLEU8, RFP2, KCNRG, ARLTS1, microRNA-15, and microRNA-16–1 [45–53]. Chromosome 12 abnormalities are represented by chromosome 12 trisomy, which induces an increased expression of genes located on this chromosome. For this disease, the sequences located between 12q13 and 12q22 are especially important. This anomaly is rarely detectable at diagnosis, but is present in more advanced stages of the disease and is also associated with more prominent aneuploidy, thus giving rise to arguments that this is an additional anomaly [54–56]. Chromosome 11 abnormalities are represented by deletions of the long arm of

Figure 1. The role of minimal residual disease in the clinical setting.

chromosome 11. One of the most important genes for this disease found in this region is ataxia telangiectasia mutated (ATM), which plays a role in DNA damage detection and induction of cell cycle arrest. Patients with this deletion present with a more aggressive form of the disease and resistance to genotoxic agents [57–60]. Chromosome 6 abnormalities are represented by deletions of the band 6q23 and are associated with abnormal morphology of the lymphocytes, higher number of prolymphocytes in the peripheral blood, higher expression of CD38 and lower survival [61–64]. Chromosome 17 abnormalities are represented by the deletion of the short arm of chromosome 17. One of the most important genes located in this region is TP53, which, along with other functions, is implicated in the DNA damage detection pathway and cell cycle arrest. Deletions of this gene are associated with resistance to first-line therapy [65–67]. This complex molecular landscape is responsible for the heterogeneity of CLL reflected in various clinical manifestations, evolution, and treatment response. This is the reason why proper follow-up methods must be used in order to better predict outcomes. Currently, together with molecular assessment of prognosis factors, MRD represents a powerful tool in predicting outcome, monitoring treatment response and decision making. Gold standard methods for MRD are PCR-based strategies using the hypervariable region of the clonal immunoglobulin heavy-chain gene and flow cytometry. Both are used in monitoring treatment response, as shown in Figure 1. Therapy for CLL is guided according to the Rai and Binet staging systems [8,68]. Another criterion in treatment choice is the presence of del (17p) or TP53 mutation [69–71].

MRD in clinical management and clinical trials MRD outcome for different therapies based on rigorously selected patient groups according to age, prognostic factors, and comorbidities is important for

CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES

appropriate decision making. There are many closed or ongoing studies regarding MRD outcome for different therapeutic regimens including fludarabine/cyclophosphamide/rituximab (FCR), bendamustine and rituximab (BR), as well as novel anti-CD20 agents. Chlorambucil is a prominent oral alkylating agent and has been used to treat CLL for the past 60 years. In recent years, it has become less popular due to availability of better and safer alternatives for fit patients, but it is still largely used in treating elder, frail patients [72–74]. Cyclophosphamide and low-dose etoposide can also be used as single agent therapies. Bendamustine was approved for CLL treatment after it has been proven to have better tolerability than fludarabine, especially in renal impaired patients [75,76]. Fludarabine is a nucleotide analog and has been in use for the past 30 years either as a single agent or in combination chemotherapy regimens. Similar to fludarabine, other nucleoside analogs used in CLL treatment with similar outcomes are cladribine and pentostatin. Cytarabine is also used especially in combination chemoimmunotherapy with oxaliplatin, fludarabine, and rituximab (OFAR regimen) in refractory disease and Richter transformation. Early chemotherapeutic combinations used in treating CLL like CVP (cyclophosphamide, vincristine, and prednisone), CMP (cyclophosphamide, melphalan, and prednisone), and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) are still used today with great efficacy [72,77–79]. Due to the improvement in single agent therapies, multiple combination regimens have been developed: fludarabine and chlorambucil, fludarabine and prednisone, chlorambucil and prednisone. Fludarabine and cyclophosphamide (FC) regimen-based studies resulted in encouraging results with a significant improvement compared to single agent complete remission (CR) rates. This combination was the first regimen that managed to overcome the negative prognostic impact of del(11p). Antibody therapy has been a major advance in the management of CLL. Currently, four different antibodies are being used in clinical practice with consistent survival rate. Alemtuzumab is a CD52-targeting monoclonal antibody extremely effective in achieving hematological and morphological remission and is active in del (17p) CLL, generally refractory to chemotherapy. A limitation of alemtuzumab is bulky lymphadenopathy, particularly lymph nodes larger than 5 cm. Toxicity-related immunosuppression and prolonged cytopenias cause significant infectious morbidity and has led to limited use of alemtuzumab by practicing physicians [80–83]. Rituximab is a CD20-targeting monoclonal antibody largely used today in

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CD20 þ lymphoid malignancies due to high efficacy, tolerability, and low toxicity in chemoimmunotherapy regimens (R-CHOP, R-CVP, R-FC, bendamustine, and rituximab) [84,85]. Chemotherapy regimens for CLL are shown in Figure 2(A–E). In a study conducted by Bottcher et al. on 493 patients, MRD was assessed on peripheral blood and bone marrow. MRD negativity was achieved significantly more in FCR-treated patients compared with FC-treated patients, in peripheral blood and in bone marrow (44% versus 28%) [4,10]. Furthermore, the predictive significance of MRD level was studied and as expected, low levels correlated with significantly longer PFS, but no difference was seen regarding overall survival (OS). MRD was also assessed in patients with mutated heavy chain immunoglobulin (IgVH) and trisomy 12, and there were no differences in outcome after 3 and 6 courses of therapy, respectively, in MRD negative patients. This observation raised the possibility of early interruption of therapy in this patient group. In long-term follow up, the progression-free survival (PFS) differed in MRD-negative patients according to IgVH mutational status – PFS at 12.8 years was 79.8% in MRDnegative patients with IgVH mutations, compared with 16.3% in MRD-negative patients without IgVH mutations. This means that treatment end-point should not be decided only on MRD status, but also on genetic risk factors [86]. For BR regimens, CLL10 trial has shown better MRD negative results at 3 months after therapy completion for FCR versus BR, but no differences at long-term follow up. Ofatumumab is a CD20-targeting monoclonal antibody with better in vitro results. Even if the half-life of ofatumumab is 21 days, its effects on B-cell depletion can last up to 7 months [87]. As in the case of rituximab, ofatumumab is administered both as a single agent therapy as well as chemoimmunotherapy in combination with well-established regimens (i.e., chlorambucil or FC regimen). Obinutuzumab is a monoclonal antibody targeted towards CD20 with a higher efficacy compared with rituximab as its structural modifications allow the antibody to bind to extracellular CD20 for a longer time period and produce significantly higher cytotoxicity. The combination of obinutuzumab and chlorambucil has proven to be effective, achieving higher CR and OS rates when compared to rituximab and chlorambucil or to other chemotherapy regimens, particularly in patients with pre-existing comorbid conditions. While obinutuzumab is well-tolerated, it has a higher incidence of infusion-related reactions compared to rituximab or ofatumumab [88]. Currently, great interest has been shown for obinutuzumab in combination with

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Figure 2. (A–E) Chemotherapy protocols for chronic lymphocytic leukemia.

other novel agents and chemoimmunotherapy regimens. Thus, BCR kinase inhibitors have provided a significant improvement in the treatment management of CLL patients due to promising efficacy and tolerability. Ibrutinib is an irreversible inhibitor of BTK and is given orally in a dose of 420 mg. Ibrutinib is generally well-tolerated and exhibits sustained improvement in CR and PR when compared with other conventional therapies. However, studies have shown higher incidence of both minor bleeding and atrial fibrillation with ibrutinib. Therefore, warfarin-based anticoagulation treatment and ibrutinib should not be concurrently administered and in the case of surgical procedures,

treatment should be held for 3–7 days before and after [89–92]. Ibrutinib does not generally result in CR, but discontinuation of treatment leads to rapid progression of disease. Thus, ibrutinib is administered continuously in responding patients until disease progression or high level of toxicity occurs. This can lead to the emergence of drug-resistance in the form of malignant cell clones [93,94]. Ibrutinib-based therapy combinations showed higher CR rates and MRD negativity achievements. There are several studies regarding ibrutinib as front-line therapy or in addition to an anti-CD20 monoclonal antibody, which may provide better results regarding MRD


negativity. However, most studies do not have a sufficient number of patients in a given subgroup [95,96]. MRD was assessed by 8-color flow cytometry in peripheral blood and bone marrow; however, due to a short follow up period, the sole conclusion was that achievement of MRD status was greater in the BR þ ibrutinib group compared to the BR group. Idelalisib is a selective PI3K-d inhibitor that promotes apoptosis of CLL B cells ex vivo Idelalisib and rituximab combination therapy was approved in 2014 for relapsed CLL with comorbid conditions otherwise not eligible for standard treatment. Toxicity usually related to elevated liver enzymes and infections occurs with prolonged continuous use [97,98]. Another molecule that shows promise is venetoclax (ABT-199), a Bcl-2-targeting agent with great promise in relapsed and refractory patients and del (17p) [99]. The phase I dose escalation study demonstrated high CR rates in patients with negative prognosis factors, which were refractory to fludarabine, had del(17p) and unmutated IgHV, even though FC-MRD protocols were not optimally standardized (4-color, site-specific methods). In the phase III study, MRD was assessed by 4- or 6-color flow cytometry in peripheral, and part of the PB MRD negative patients also had bone marrow assessment. Taken into consideration that inclusion criteria were 17p deletion, relapsed or refractory disease, 18 of 45 patients showed MRD negativity for over 8.8 months in peripheral blood, and six of 10 patients also presented with MRD negativity in bone marrow. MRD was also assessed for different combinations, as is the case of venetoclax plus rituximab or venetoclax plus obinutuzumab and ibrutinib. A report of the British Society of Hematology regarding the role of MRD in clinical trials and observations concluded that MRD negativity correlates with PFS and OS in an independent manner, regardless of disease stage or line of treatment. Still, the most important impact of MRD achievement is reported for the ones that have received frontline therapy. They have a 10year PFS of 65% versus 10%, as well as 1-year OS of 70% versus 30% for MRD-negative cases in comparison with MRD-positive cases, respectively [100]. The International Workshop on CLL recommended the assessment of MRD in clinical trials, and both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have considered MRD as a potential endpoint for clinical trials. The EMA developed a guideline for use of MRD as an intermediate endpoint in CLL [35]. In the US, the presence or absence of MRD predicted PFS and, to a lesser degree, OS among CLL patients who achieved complete or partial remission with six courses of chemoimmunotherapy, based on

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two randomized phase III trials. Kovacs et al. [101] examined the predictive value of measuring MRD in peripheral blood by using 4-color flow cytometry at a threshold of 104. Data were analyzed from 554 adult CLL patients who achieved complete or partial remission during two phase III trials – one (CLL8) comparing fludarabine and cyclophosphamide to fludarabine, cyclophosphamide, and rituximab (FCR) and a second (CLL10) comparing FCR to bendamustine and rituximab [101]. The median PFS at the end of treatment was 61 months for the 34% (186 patients) who attained MRD-negative CR, 54 months for the 29% of patients who attained MRD-negative partial remission (PR), 35 months for the 7% of patients who attained MRDpositive CR, and 21 months for the 30% of patients who attained MRD-positive PR. PFS did not differ significantly between MRD-negative CR and MRD-negative PR patients, but was significantly longer for MRD-negative PR patients than for MRD-positive CR patients (p ¼ .048). PFS was even more distinct for MRD-positive CR patients compared to those who attained MRD-positive PR (p ¼ .002). OS was significantly shorter only when patients had MRD-positive PR rather than MRD-negative CR (72 months versus not reached, p ¼ .001). Among the MRD-negative PR patients, 16% had only residual lymphadenopathy, 11% had only bone marrow involvement, 48% had only enlarged spleen volume, and 25% had more than one organ system affected, the researchers noted. Importantly, PFS for MRD-negative PR with residual splenomegaly (63 months) was similar to that for MRD-negative CR (61 months, p ¼ .354). In contrast, patients with MRD-negative PR and residual lymphadenopathy had shorter PFS than did MRD-negative CR patients (31 months, p ¼ .001). Hematopoietic stem cell transplantation (HSCT) has become an increasingly less used treatment approach in CLL due in part to the fact that transplantation conditioning high-dose chemotherapy has proven to be largely inefficient in del (17p) CLL. Autologous transplantation does not offer a superior OS when compared with non-transplanted patients. Allogeneic SCT does present a potential curative treatment option. Still, in recent years, with the increased availability of novel single-agent therapies with better tolerance and lower toxicity and the absence of chronic graft-versus-host disease, HSCT in CLL has become a less popular approach [102,103]. MRD detection in post-transplantation CLL provides different information according to the type of transplant and the time of investigation. Currently, it is believed that kinetics of MRD and long-term follow up is more important than a single post-transplant evaluation. Generally, it is a recognized fact that long-term

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MRD negativity is more frequent in allogenic (allo)-HSCT compared with autologous HSCT [104]. More recent approaches in allo-HSCT with reduced conditioning and the recognition of the important role of graft-versusleukemia effect has changed the way MRD is monitored in these cases. This means that a long-term follow up is recommended [105,106], even when MRD negativity appears late after transplantation, is attributed to GVL and can be further consolidated with donor lymphocyte infusion [107–109]. Local radiation is a wildly used approach in treating locally symptomatic lymphadenopathy and symptomatic splenomegaly but with limited and shortlived efficacy requiring concurrent standard treatment [110–112]. Anti CD-19 autologous CAR T-cell therapy is a promising approach for many hematological malignancies of B-cell origin and was also used in some cases of relapsed and refractory CLL, generating optimistic results in short-term follow up for MRD negativity [113]. As evidence from the studies presented above regarding MRD assessment in the clinical settings and clinical trials are not uniform, results can hardly be compared. CLL is clinically heterogeneous, especially in regard to prognosis, therapy regimens are very different, and patients groups differ not only by age and sex but also by comorbidities and particular responses to therapeutic agents. The use of MRD as a treatment endpoint needs further clarification after consensus guidelines regarding investigation protocols (standardized methods and clearly defined time points) are universally applied. According to 2008 CLL guidelines, MRD should be investigated 3 months and further after therapy completion, and for targeted antibody treatment, bone marrow should be analyzed. Negative MRD in peripheral blood does not always mean negativity in bone marrow and any of these statuses can change over time, so repeated testing is required. Although a lot of work has been done regarding MRD, definitive conclusions regarding therapy endpoint are not yet available. CLL MRD use in clinical practice still raises questions and concerns. The study of Strati et al. opens the possibility of safe discontinuation of treatment after FC-MRD achievement, even before completion of standard chemoimmunotherapy, especially for patients with comorbidities and treatment toxicity. Consequently, as physicians consider more precise follow-up, assessing treatment endpoint by MRD could become an option for clinical decision making in the future, together with treatment compliance, pharmacogenetics, renal and/or hepatic impairment. The goal is to achieve optimal response and concomitantly minimize toxic side effects [114]. More and more data suggests that MRD

Figure 3. The protocol for the diagnosis and follow-up of chronic lymphocytic leukemia.

achievement, demonstrated by FC and/or PCR, can be used as a treatment endpoint in clinical trials, but also in chemoimmunotherapy [115].

Minimal residual disease (MRD) in CLL: flow-cytometry-based approach MRD is considered to be the level of disease that can be detectable by sensitive techniques and shows an incomplete treatment and a probability of disease relapse [116], as shown in Figure 3. MRD detection has been continuously improved by the quick development of both flow cytometry and molecular biology technology, as well as by next generation sequencing (NGS). PCR and other similar assays have the advantage of indicating specific transcripts of interest or, in the case of genomic techniques, have the advantage of assessing gene modifications that are not expressed. In the latter mentioned direction, studies on silenced, but present, BCR-ABL fusion gene have been made [117,118]. Flow cytometry is used to detect the immunophenotype characteristic of malignant cells, but this assay may not be available in all laboratories, because of the requirement to detect one leukemic cell in 104 normal leukocytes. In recent years, NGS has been tested for detecting MRD, with promising results [119–123]. The advantages of the latter mentioned technique include the reduction of false negatives by screening for a larger number of clones [124,125]. Thus, the role of detecting MRD is to help make therapeutic decisions regarding whether continuation of a treatment is appropriate. Flow cytometry is used to detect surface or intracellular antigens of CLL cells and describe their immunophenotype. The classic characteristics of CLL cells are positivity for CD19, CD5, CD23, CD22, CD79b low/negative, CD43, CD200; negativity for CD10, FMC7, and CD103 and low expression of CD20 and surface


immunoglobulins. The first attempt to detect MRD in CLL used the same principles as for CLL disease detection through 2-color flow cytometry. The problem with this approach was the lack of sensitivity, being able to detect one CLL cell in 200 normal cells and the immunophenotype is not unique to the CLL cells [126]. A 3color flow cytometer will enable the detection of CLL cells by expression of CD19, CD5 and is not appropriate to use for the detection of MRD, where it is considered that the minimal sensitivity is 104 [127–130]. To surpass this drawback, Rawson et al. developed a 4-color flow cytometry method that reached the sensitivity of 104 cells. It has been proven that this protocol is able to discriminate between CLL cells and the normal B and T lymphocyte populations. Also, another advantage of this technique is that it does not require custom reagents for each patient. Over 50 CLL-specific antibody combinations were tested in order to identify a small number of efficient combinations which showed a high degree of correlation with RQ-ASO and IgH-PCR and low inter-laboratory variations. [131,132]. The downside of flow cytometry is that the interpretation of the result is operator dependent and there have to be enough events acquired so that the sensitivity reaches the minimum of 104. Thus, a hypocellular sample will be inappropriate for MRD detection [133]. Still, in blood samples with few lymphocytes, as is the case of acute infections where neutrophils are more abundant, the total amount of blood used should be slightly higher in order to reach an adequate sensitivity. That is why further attempts were made by using more colors, in order to achieve higher sensitivity with fewer antigens, fewer cells and a simplified analysis algorithm. Flow cytometry-based assays first provided 8- and 10color flow cytometry methods and the latter achieved a sensitivity of 105, with the condition that the minimal number of cells acquired has to be 1.8 106 [133–135]. The methods used for higher sensitivity in MRD determination were introduced by Rawstron et al. A panel of antibodies was devised for this very assessment: CD5, CD19, CD20, CD79b, at which, CD38 was later introduced for the better discrimination of normal bone marrow progenitors from CLL cells [132]. Intermediate protocols were for 6-color cytometers [133] using CD19/CD5/CD20 with CD3/CD38/CD79b and CD81/CD22/CD43, and these showed good correlation and linearity when compared with the previous 4-color approach at higher sensitivity levels. Using more colors proved to be more cost efficient, less time consuming and simpler than previous FC methods and molecular methods [134]. The 8-color panels use a core combination of the same markers and offer the possibility to add supplementary markers to increase sensitivity up

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to 106, as demonstrated by comparison with highthroughput sequencing using the ClonoSEQ assay [35] However, because of the depletion of CD20 positive cells in patients treated with rituximab or other antiCD20 antibodies (ofatumumab and obinutuzumab), antibody panels using CD20 were deemed inappropriate for these patients. Thus, a new antibody panel had to be developed, which contained: CD81, CD22, CD5, and CD19, as described by the same group from Leeds, UK [136]. Another important aspect related to therapy is the moment that MRD assessment is made, for example, after alemtuzumab treatment has been administered. Bone marrow CLL cells do not present a significant decrease, but they are depleted from the peripheral blood, so an assessment of the peripheral blood immediately after the treatment may therefore yield a false negative result, as shown by Monserrat et al. [106]. Nevertheless, because of the operatordependent results in the case of flow cytometry, an international standardization of CLL MRD assessment has been developed by a set of laboratories under the supervision of the European Research Initiative on CLL (ERIC) [35,134]. Concomitant studies were made in order to achieve specificity and sensitivity in single tubes by 8- or 10color cytometers, by adding specific markers such as CD160 [137–139]. One of the main assessments that resulted was that flow MRD can detect one CLL cell in 104 leukocytes. Peripheral blood can be used in most cases for MRD detection, with the exception of the case when the patient is under antibody therapy, in which case, bone marrow aspirate should be used. Flow cytometry presents a high enough sensitivity, low-cost and quick result generation to be used in routine clinical practice. For a more sensitive result and if MRD eradication is wanted for the selected patient, then allele-specific PCR should be used [121]. However, recent flow cytometry methods provide a sensitivity up to one in a million [35]. The relevance of prediction role of MRD is shown in Table 1, showing the survival benefit of both flow and molecular MRD negative patients. Multiple studies using multivariate analysis show that FC-MRD represents an independent prognostic factor for OS and PFS, when compared with different prognostic factors, such as cytogenetic aberrations, ZAP70, IgVH mutational status [86], but also with more recently discovered mutations in TP53, NOTCH1, MYD88, and SF3B [149]. Flow cytometry is currently widely available and creation of inter-institutional platforms for a uniform investigation of MRD should be a future goal. Implementation of standard operating procedures agreed by all institutions implied in patients care and

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Table 1. Minimal residual disease in the clinical setting for chronic lymphocytic leukemia. The protocol described in the presented studies are based on flow cytometry, either 3-color or 4-color, or on PCR. PCR is carried out for IgH or it is based on allele-specific oligonucleotide PCR (ASO-PCR). Author/institution Robertson et al./MD Anderson Cancer Center, Houston, USA Short et al./MD Anderson Cancer Center, Houston, USA Montillo et al./Ospedale Niguarda Ca' Granda, Milano, Italy Del Poeta et al./University Tor Vergata, S. Eugenio Hospital, Rome, Italy Wendtner et al./Ludwig-Maximilians-University, Munich, Germany Bosch et al./Hospital Clınic, Villarroel, Barcelona, Spain Moreton et al./Leeds Teaching Hospitals, NHS Trust, Leeds, United Kingdom Strati et al./MD Anderson Cancer Center, Houston, USA Wierda et al./MD Anderson Cancer Center, Houston, USA Rawstron et al./St James's Institute of Oncology, Leeds, United Kingdom Moreno et al./Hospital Clınic, Villarroel, Barcelona, Spain Moreno et al./Hospital Clınic, Villarroel, Barcelona, Spain Ritgen et al./University of Schleswig-Holstein, Kiel, German Caballero et al./Hospital Universitario Salamanca (CAUSA/IBSAL), Salamanca, Spain

Disease status Unselected Therapy-naïve Therapy-naïve Therapy-naïve Therapy-naïve Relapsed/refractory Relapsed/refractory Relapsed/refractory Relapsed/refractory Therapy-naive Relapsed/refractory Relapsed/refractory Relapsed/refractory Relapsed/refractory

MRD assessment protocol Flow for CD19/CD5 Flow for CD19/CD5 PCR for IgH 3-colour flow ASO-PCR 4-colour flow 4-colour flow ASO-PCR ASO-PCR PCR for IgH PCR for IgH 4-colour flow ASO-PCR 4-colour flow

Reference [127] [140] [141] [142] [143] [144] [145] [114] [146] [35] [106] [147] [108] [148]

Figure 4. (A–C) Templates for the acquisition of CLL cells.

clinical trials will strengthen the quality of provided data and provide comparable information [35]. PCRbased testing for MRD in CLL includes several methods, including consensus, nested, and allele-specific oligonucleotide (ASO) immunoglobulin heavy chain.

MRD in CLL: NGS-based approach As MRD detection continues to move from research to clinical practice, where it can be an independent prognostic marker, the methodologies by which MRD is studied have to develop further so they can suit the needs of clinical practice. An emerging technology used for MRD detection is NGS. The advantage of NGS is that it uses the same primers regardless of the patient, compared with allele-specific oligonucleotide PCR, where patient customization is necessary. The primers used for NGS target the IGH VDJ regions of immunoglobulins and assess the clonality of the analyzed population [150]. Logan et al. showed not only that by using 454 pyrosequencing IGH-HTS, the sensitivity of MRD detection can reach values from 105 to 106, but also that this technology, due to the fact that patient customization is not necessary, can be applied in a clinical context [151,152]. One of the most important applications for

CLL pointed out by the Stanford group was the evaluation of MRD after hematopoietic stem cell transplantation. Other applications of this technology are considered to be the assessment of new therapies in treating CLL and the follow-up of immune reconstitution after allo-HCT. One drawback of this assay appears when the CLL clone presents mutations in the primer’s annealing sites. Campbell et al. [153] showed that it is linked to the presence of oligoclonal mutant lymphocytes. By using this protocol, it was shown that the use of rituximab prophylaxis for GVHD had less mutations in the IGH when compared to patients that did not receive additional therapy [154]. The use of this assessment is sustained by the fact that after HCT, CLL relapse is still common. Thus, a sensitive method which can be widely used for patients that undergo a HCT to predict the prognostics of transplanted patients and to help decide the therapeutic approach is still needed [147,155,156]. Nevertheless, assays such as the NGS and PCR-based ones have several limitations, as somatic hypermutations process in Ig genes further hampers primer annealing in mature B-cell malignancies [150,157]. As a proof-of-concept for the presented theoretical knowledge, we present the case of a patient diagnosed


Figure 5. (A–D) Templates for the analysis of CLL cells.

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Figure 6. (A–C) A representative example of CLL MRD acquisition.

with CLL for which MRD assessment was done by flow cytometry. The templates used for acquisition are presented in Figure 4(A–C). For the building of the analysis template (Figure 5(A–D)), the protocol first starts with eliminating the doublets from the singlets window. Afterwards, leukocytes are selected by selecting CD45 þ cells on the SSC/CD45 histogram and improving the purity of the cell population after having eliminated the doublets, represented by SING45. Lymphocytes are selected based on morphometry on the SSC/CD45 histogram and a pure population is selected, entitled SING LY by crossing the histograms: CE45, SINGLETS, LY TOT, and SING LY. In the last cell population, all the B cells are selected, on the CD19/CD20 histogram, before the CD19 and CD19low sub-populations are selected on the SSC/CD19 histogram. Based on CD19 events, the physician is able to create gates that allow the selection of the population of interest using the specific markers available for flow cytometry. The CLL population is

represented by the crossing-over of all these histograms. T lymphocytes are selected based on their CD43 þ CD5 þ high expression. A representative example of CLL MRD acquisition is presented in Figure 6(A–C). In a second step, an acquisition restricted on the fluorescence threshold for CD5 (measured on T and B lymphocytes) and CD19 (measured on B-lymphocytes) is performed, as shown in Figure 7(A–C). This step is required in order to improve the sensitivity through an increase in the number of events of interest by removing the granulocytes, most of the monocytes, debris, erythrocytes, and nucleated cells of the bone marrow. Considering that several studies have shown the influence of CLL MRD detection for a patient’s prognosis and therapeutic approach, it is of great importance that widely available technologies are available for this assessment. MRD detection is slowly evolving from bench to bedside. The introduction of this new assessment in clinical practice will bring us a step forward in


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Figure 7. (A–C) Example of acquisition restricted on the fluorescence threshold for CD5 (measured on T and B lymphocytes) and CD19 (measured on B-lymphocytes).

the way of personalized medicine and better prediction of disease evolution.

Conclusions MRD refers to the evaluation of the residual leukemia cells in patients after treatment. The threshold for MRD negativity is set to less than 1:100,000 malignant cells detected in mononucleated cells. This threshold is set taking into consideration the currently available techniques for detecting MRD. MRD evaluation is considered to be the best predictor for evaluation of relapse in hematological malignancies and is now used in different studies and trials for evaluating the effect of different treatments. There are two main techniques implemented in MRD evaluation: flow cytometry and PCR. Flow cytometry is mainly used for detecting a cell with a specific phenotype among the normal cell population, whereas PCR methods are used to amplify the immunoglobulin heavy chain variable region of patient DNA. One method that can overcome this problem and

have an even higher sensitivity than flow cytometry and PCR methods is NGS. NGS is defined as a new sequencing method that overcomes the limitation of Sanger sequencing and allows the use of low sample quantity, multiplexing samples, and sequencing several targets at once. Also, it confers better results at a lower cost and less time. Over time, several different NGS platforms have been developed and this method has gained interest in both research and clinical settings because by applying this modern protocol, the relapse or response to treatment of patients can be detected sooner, improving patient outcomes.

Acknowledgements Ciprian Tomuleasa, Cristina Selicean, and Sonia Cismas contributed equally to the current paper.

Disclosure statement No potential conflict of interest was reported by the authors.

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Funding

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Ciprian Tomuleasa received funding from the Romanian Research Ministry, contracts [PN-II-RU-TE-2014-4-1783] (awarded to Young Research Teams) and [CNFIS-FDI2017–1350] (awarded to Institutional development funds), as well as from an international collaboration grant between Romania and PR China, contract [57 BM/2016].

[16]

References [1]

[2] [3]

[4]

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

A V. Sur la resorption du pusuaet sur l'alteration du sang dans les maladies clinique de persection nenemant. Premier observation. Rev Med. 1827;2:216. R V. Weisses Blut und Milztumoren. I Med Z. 1846;5:157. Rubin AD, Schultz E. Surface immunoglobulins on lymphocytes in leukemia. N Engl J Med. 1972;287: 989–990. Fialkow PJ, Najfeld V, Reddy AL, et al. Chronic lymphocytic leukaemia: clonal origin in a committed B-lymphocyte progenitor. Lancet. 1978;2:444–446. Salsano F, Froland SS, Natvig JB, et al. Same idiotype of B-lymphocyte membrane IgD and IgM. Formal evidence for monoclonality of chronic lymphocytic leukemia cells. Scand J Immunol. 1974;3:841–846. Fu SM, Winchester RJ, Feizi T, et al. Idiotypic specificity of surface immunoglobulin and the maturation of leukemic bone-marrow-derived lymphocytes. Proc Natl Acad Sci USA. 1974;71:4487–4490. Schroer KR, Briles DE, Van Boxel JA, et al. Idiotypic uniformity of cell surface immunoglobulin in chronic lymphocytic leukeima. Evidence for monoclonal proliferation. J Exp Med. 1974;140:1416–1420. Rai KR, Sawitsky A, Cronkite EP, et al. Clinical staging of chronic lymphocytic leukemia. Blood. 1975;46: 219–234. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2007. CA: Cancer J Clin. 2007;57:43–66. Torre LA, Siegel RL, Ward EM, et al. Global cancer incidence and mortality rates and trends–an update. Cancer Epidemiol. Biomarkers Prev.: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 2016;25:16–27. Mortality GBD, Causes of Death C. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388: 1459–1544. Cartwright RA, Gurney KA, Moorman AV. Sex ratios and the risks of haematological malignancies. Br J Haematol. 2002;118:1071–1077. Dores GM, Anderson WF, Curtis RE, et al. Chronic lymphocytic leukaemia and small lymphocytic lymphoma: overview of the descriptive epidemiology. Br J Haematol. 2007;139:809–819. Molica S. Sex differences in incidence and outcome of chronic lymphocytic leukemia patients. Leuk Lymphoma. 2006;47:1477–1480.

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

Cerhan JR, Vachon CM, Habermann TM, et al. Hormone replacement therapy and risk of nonHodgkin lymphoma and chronic lymphocytic leukemia. Cancer Epidemiol. Biomarkers Prev.: A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 2002;11:1466–1471. Tamura K, Sawada H, Izumi Y, et al. Chronic lymphocytic leukemia (CLL) is rare, but the proportion of T-CLL is high in Japan. Eur J Haematol. 2001;67: 152–157. Yuille MR, Matutes E, Marossy A, et al. Familial chronic lymphocytic leukaemia: a survey and review of published studies. Br J Haematol. 2000;109: 794–799. Scarfo L, Ghia P. What does it mean I have a monoclonal B-cell lymphocytosis?: recent insights and new challenges. Semin Oncol. 2016;43:201–208. Scarfo L, Ferreri AJ, Ghia P. Chronic lymphocytic leukaemia. Crit Rev Oncol/Hematol. 2016;104:169–182. Rawstron AC, Bennett FL, O'Connor SJ, et al. Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med. 2008;359: 575–583. Rawstron AC, Yuille MR, Fuller J, et al. Inherited predisposition to CLL is detectable as subclinical monoclonal B-lymphocyte expansion. Blood. 2002;100: 2289–2290. Nieto WG, Almeida J, Romero A, et al. Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach. Blood. 2009;114:33–37. Marti G, Abbasi F, Raveche E, et al. Overview of monoclonal B-cell lymphocytosis. Br J Haematol. 2007;139:701–708. Wierda WG, Zelenetz AD, Gordon LI, et al. NCCN Guidelines Insights: chronic lymphocytic leukemia/ small lymphocytic leukemia, version 1.2017. J Natl Compr Canc Netw. 2017;15:293–311. Kravic-Stevovic T, Bogdanovic A, Boskovic D, et al. Ribosome-lamella complexes in the peripheral blood of patients with chronic lymphocytic leukemia are associated with serological immune deficiency. Ultrastruct Pathol. 2010;34:31–34. Lee S, Graham LM, Chan G, et al. A diagnostic mystery solved by electron microscopy: a case of an "atypical" lymphoproliferative disorder. Ultrastruct Pathol. 2012;36:362–365. Nagy-Simon T, Tatar AS, Craciun AM, et al. Antibody conjugated, raman tagged hollow gold-silver nanospheres for specific targeting and multimodal DarkField/SERS/Two Photon-FLIM imaging of CD19(þ) B Lymphoblasts. ACS Appl Mater Interfaces. 2017;9: 21155–21168. Thakral B, Konoplev S. "Soccer ball" cells to "donut" cells: an unusual case of Richter syndrome. Blood. 2017;130:2358. Quesada AE, Konoplev S. Classical Hodgkin lymphoma arising in a patient with chronic lymphocytic leukemia (Richter syndrome). Blood. 2017;130:2151.


[30]

[31]

[32]

[33]

[34] [35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

Jamroziak K, Tadmor T, Robak T, et al. Richter syndrome in chronic lymphocytic leukemia: updates on biology, clinical features and therapy. Leuk Lymphoma. 2015;56:1949–1958. Baliakas P, Kanellis G, Stavroyianni N, et al. The role of bone marrow biopsy examination at diagnosis of chronic lymphocytic leukemia: a reappraisal. Leuk Lymphoma. 2013;54:2377–2384. Sah SP, Matutes E, Wotherspoon AC, et al. A comparison of flow cytometry, bone marrow biopsy, and bone marrow aspirates in the detection of lymphoid infiltration in B cell disorders. J Clin Pathol. 2003;56:129–132. Dezorella N, Kay S, Baron S, et al. Measurement of lymphocyte aggregation by flow cytometry-physiological implications in chronic lymphocytic leukemia. Cytometry. 2016;90:257–266. Ozet M. Immunophenotyping of chronic lymphocytic leukemia. Clin Lab. 2017;63:1621–1626. Rawstron AC, Fazi C, Agathangelidis A, et al. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study. Leukemia. 2016;30:929–936. Tees MT, Flinn IW. Chronic lymphocytic leukemia and small lymphocytic lymphoma: two faces of the same disease. Expert Rev Hematol. 2017;10:137–146. Craig FE. The utility of peripheral blood smear review for identifying specimens for flow cytometric immunophenotyping. Int J Lab Hematol. 2017;39:41–46. Sandes AF, de Lourdes Chauffaille M, Oliveira CR, et al. CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry. Cytometry B Clin Cytom. 2014;86:98–105. Kohnke T, Wittmann VK, Bucklein VL, et al. Diagnosis of CLL revisited: increased specificity by a modified five-marker scoring system including CD200. Br J Haematol. 2017;179:480–487. Palumbo GA, Parrinello N, Fargione G, et al. CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia. Leuk Res. 2009;33:1212–1216. Challagundla P, Medeiros LJ, Kanagal-Shamanna R, et al. Differential expression of CD200 in B-cell neoplasms by flow cytometry can assist in diagnosis, subclassification, and bone marrow staging. Am J Clin Pathol. 2014;142:837–844. Rahman K, Kumar P, Gupta R, et al. Role of CD200 in differential diagnosis of mature B-cell neoplasm. Int J Lab Hematol. 2017;39:384–391. Rawstron AC, Kreuzer KA, Soosapilla A, et al. Reproducible diagnosis of Chronic Lymphocytic Leukemia by flow cytometry: an European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) harmonisation project. Cytometry Part B, Clinical Cytometry. 2018;94: 121–128. Baskar S, Kwong KY, Hofer T, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

[58]

341

Cancer Res: Off J Am Assoc Cancer Res. 2008;14: 396–404. Dohner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910–1916. Put N, Konings P, Rack K, et al. Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: a Belgian multicentric study. Genes Chromosom Cancer. 2009;48:843–853. Vargova K, Pesta M, Obrtlikova P, et al. MiR-155/miR150 network regulates progression through the disease phases of chronic lymphocytic leukemia. Blood Cancer J. 2017;7:e585. Gonzalez-Gascon YMI, Hernandez-Sanchez M, Rodriguez-Vicente AE, et al. Characterizing patients with multiple chromosomal aberrations detected by FISH in chronic lymphocytic leukemia. Leuk Lymphoma. 2018;59:633–642. Murphy EJ, Neuberg DS, Rassenti LZ, et al. Leukemiacell proliferation and disease progression in patients with early stage chronic lymphocytic leukemia. Leukemia. 2017;31:1348–1354. Kim JA, Hwang B, Park SN, et al. Genomic profile of chronic lymphocytic leukemia in Korea identified by targeted sequencing. PLoS One. 2016;11:e0167641. Nguyen-Khac F, Borie C, Callet-Bauchu E, et al. Cytogenetics in the management of chronic lymphocytic leukemia: an update by the Groupe francophone de cytogenetique hematologique (GFCH). Ann Biol Clin. 2016;74:561–567. Dighiero G, Hamblin TJ. Chronic lymphocytic leukaemia. Lancet. 2008;371:1017–1029. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 2002;99: 15524–15529. Sava GP, Speedy HE, Di Bernardo MC, et al. Common variation at 12q24.13 (OAS3) influences chronic lymphocytic leukemia risk. Leukemia. 2015;29: 748–751. Hui H, Fuller KA, Chuah H, et al. Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia. Methods. 2018;134–135: 32–40. Gonzalez-Gascon YMI, Hernandez-Sanchez M, Rodriguez-Vicente AE, et al. A high proportion of cells carrying trisomy 12 is associated with a worse outcome in patients with chronic lymphocytic leukemia. Hematol Oncol. 2016;34:84–92. Skowronska A, Austen B, Powell JE, et al. ATM germline heterozygosity does not play a role in chronic lymphocytic leukemia initiation but influences rapid disease progression through loss of the remaining ATM allele. Haematologica. 2012;97:142–146. Marasca R, Maffei R, Martinelli S, et al. Clinical heterogeneity of de novo 11q deletion chronic lymphocytic leukaemia: prognostic relevance of extent of 11q deleted nuclei inside leukemic clone. Hematol Oncol. 2013;31:88–95.

342

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

C. TOMULEASA ET AL.

Dohner H, Stilgenbauer S, James MR, et al. 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood. 1997;89:2516–2522. Austen B, Skowronska A, Baker C, et al. Mutation status of the residual ATM allele is an important determinant of the cellular response to chemotherapy and survival in patients with chronic lymphocytic leukemia containing an 11q deletion. J Clin Oncol. 2007;25:5448–5457. Slager SL, Rabe KG, Achenbach SJ, et al. Genomewide association study identifies a novel susceptibility locus at 6p21.3 among familial CLL. Blood. 2011;117:1911–1916. Wang DM, Miao KR, Fan L, et al. Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia. Leuk Lymphoma. 2011;52:230–237. Vladareanu AM, Ciufu C, Neagu AM, et al. The impact of hepatitis viruses on chronic lymphoproliferative disorders – preliminary results. J Med Life. 2010;3:320–329. Vasile D, Vladareanu AM, Bumbea H. Peripheral T and NK cell non-hodgkin lymphoma a challenge for diagnosis. Maedica (Buchar). 2014;9:104–108. Chapiro E, Lesty C, Gabillaud C, et al. "Double-hit" chronic lymphocytic leukemia: an aggressive subgroup with 17p deletion and 8q24 gain. Am J Hematol. 2018;93:375–382. Barrientos JC. Sequencing of chronic lymphocytic leukemia therapies. Hematol Am Soc Hematol Educ Program. 2016;2016:128–136. Rossi D, Terzi-di-Bergamo L, De Paoli L, et al. Molecular prediction of durable remission after firstline fludarabine-cyclophosphamide-rituximab in chronic lymphocytic leukemia. Blood. 2015;126: 1921–1924. Binet JL, Auquier A, Dighiero G, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer. 1981;48:198–206. Rai KR, Peterson BL, Appelbaum FR, et al. Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. N Engl J Med. 2000;343:1750–1757. Keating MJ, O'Brien S, Albitar M, et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia. J Clin Oncol. 2005;23:4079–4088. Byrd JC, Brown JR, O'Brien S, et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014;371:213–223. Hallek M. Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment. Am J Hematol. 2017;92:946–965. Innocenti I, Autore F, Pasquale R, et al. Treatment approach for elderly and unfit patients with chronic lymphocytic leukemia. Expert Rev Hematol. 2017;10: 1069–1076. Chanan-Khan AA, Zaritskey A, Egyed M, et al. Lenalidomide maintenance therapy in previously

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

treated chronic lymphocytic leukaemia (CONTINUUM): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Haematol. 2017;4:e534–e543. Wilkes GM. Drug essentials. An alkylating agent for CLL and NHL. Oncology (Williston Park, NY). 2009;23:55–56. Knauf W. Bendamustine in the treatment of chronic lymphocytic leukemia. Expert Rev Anticancer Ther. 2009;9:165–174. Mauro FR, Carella AM, Molica S, et al. Fludarabine, cyclophosphamide and lenalidomide in patients with relapsed/refractory chronic lymphocytic leukemia. A multicenter phase I-II GIMEMA trial. Leuk Lymphoma. 2017;58:1640–1647. Howard DR, Munir T, McParland L, et al. Clinical effectiveness and cost-effectiveness results from the randomised, Phase IIB trial in previously untreated patients with chronic lymphocytic leukaemia to compare fludarabine, cyclophosphamide and rituximab with fludarabine, cyclophosphamide, mitoxantrone and low-dose rituximab: the Attenuated dose Rituximab with ChemoTherapy In Chronic lymphocytic leukaemia (ARCTIC) trial. Health Technol Assess. 2017;21:1–374. Wendtner CM, Gregor M. Current perspectives on the role of chemotherapy in chronic lymphocytic leukemia. Leuk Lymphoma 2018;59:300–310. Voorhies BN, Stephens DM. What is optimal front-line therapy for chronic lymphocytic leukemia in 2017? Curr Treat Options Oncol. 2017;18:12. Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015;526:525–530. Al-Sawaf O, Fischer K, Herling CD, et al. Alemtuzumab consolidation in chronic lymphocytic leukaemia: a phase I/II multicentre trial. Eur J Haematol. 2017;98:254–262. Schweighofer CD, Ritgen M, Eichhorst BF, et al. Consolidation with alemtuzumab improves progression-free survival in patients with chronic lymphocytic leukaemia (CLL) in first remission: long-term follow-up of a randomized phase III trial of the German CLL Study Group (GCLLSG). Br J Haematol. 2009;144:95–98. Farahani M, Bokharaei-Salim F, Ghane M, et al. Prevalence of occult hepatitis C virus infection in Iranian patients with lymphoproliferative disorders. J Med Virol. 2013;85:235–240. Coppola N, Pisaturo M, Guastafierro S, et al. Increased hepatitis C viral load and reactivation of liver disease in HCV RNA-positive patients with oncohaematological disease undergoing chemotherapy. Digestive Liver Disease: Official Journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2012;44:49–54. Bottcher S, Ritgen M, Fischer K, et al. Minimal residual disease quantification is an independent predictor of progression-free and overall survival in chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG CLL8 trial. J Clin Oncol. 2012;30:980–988.


[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

Lundin J, Porwit-MacDonald A, Rossmann ED, et al. Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for Bcell chronic lymphocytic leukaemia. Leukemia. 2004;18:484–490. Herter S, Herting F, Mundigl O, et al. Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and of atumumab in vitro and in xenograft models. Mol Cancer Ther. 2013;12:2031–2042. Thompson PA, Burger JA. Bruton's tyrosine kinase inhibitors: first and second generation agents for patients with Chronic Lymphocytic Leukemia (CLL). Expert Opin Investig Drugs. 2018;27:31–42. Ayed AO, Parikh SA. Management of patients with chronic lymphocytic leukemia at high risk of relapse on ibrutinib therapy. Leuk Lymphoma. 2017;1–10. Vladareanu AM, Cisleanu D, Dervesteanu M, et al. Myeloma cells with asurophilic granules–an unusual morphological variant – case presentation. J Med Life. 2008;1:74–86. Bagacean C, Tempescul A, Patiu M, et al. Atypical aleukemic presentation of large granular lymphocytic leukemia: a case report. Ott. 2017;10:31–34. Awan FT, Byrd JC. New strategies in chronic lymphocytic leukemia: shifting treatment paradigms. Clin Cancer Res: Off J Am Assoc Cancer Res. 2014;20:5869–5874. Collett L, Howard DR, Munir T, et al. Assessment of ibrutinib plus rituximab in front-line CLL (FLAIR trial): study protocol for a phase III randomised controlled trial. Trials. 2017;18:387. Jain P, Keating MJ, Wierda WG, et al. Long-term follow-up of treatment with ibrutinib and rituximab in patients with high-risk chronic lymphocytic leukemia. Clin Cancer Res. 2017;23:2154–2158. Chanan-Khan A, Cramer P, Demirkan F, et al. Ibrutinib combined with bendamustine and rituximab compared with placebo, bendamustine, and rituximab for previously treated chronic lymphocytic leukaemia or small lymphocytic lymphoma (HELIOS): a randomised, double-blind, phase 3 study. Lancet Oncol. 2016;17:200–211. Waldron M, Winter A, Hill BT. Pharmacokinetic and pharmacodynamic considerations in the treatment of chronic lymphocytic leukemia: ibrutinib, idelalisib, and venetoclax. Clin Pharmacokinet. 2017;56: 1255–1266. Jones JA, Robak T, Brown JR, et al. Efficacy and safety of idelalisib in combination with ofatumumab for previously treated chronic lymphocytic leukaemia: an open-label, randomised phase 3 trial. Lancet Haematol. 2017;4:e114–e126. Slinger E, Thijssen R, Kater AP, et al. Targeting antigen-independent proliferation in chronic lymphocytic leukemia through differential kinase inhibition. Leukemia. 2017;31:2601–2607. Kwok M, Rawstron AC, Varghese A, et al. Minimal residual disease is an independent predictor for 10-year survival in CLL. Blood. 2016;128:2770–2773.

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

343

Kovacs G, Robrecht S, Fink AM, et al. Minimal residual disease assessment improves prediction of outcome in patients with ChronicLymphocytic Leukemia (CLL) who achieve partial response: comprehensive analysis of two phase III studies of the German CLL Study Group. J Clin Oncol. 2016;34:3758–3765. Schwarzbich MA, McClanahan F, Gribben J. Allogeneic transplantation for chronic lymphocytic leukemia in the age of novel treatment strategies. Oncology. 2016;30:526–533, 540. Kharfan-Dabaja MA, El-Asmar J, Awan FT, et al. Current state of hematopoietic cell transplantation in CLL as smart therapies emerge. Best Pract Res Clin Haematol. 2016;29:54–66. Esteve J, Villamor N, Colomer D, et al. Stem cell transplantation for chronic lymphocytic leukemia: different outcome after autologous and allogeneic transplantation and correlation with minimal residual disease status. Leukemia. 2001;15:445–451. Dreger P, Dohner H, Ritgen M, et al. Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial. Blood. 2010;116:2438–2447. Moreno C, Villamor N, Colomer D, et al. Clinical significance of minimal residual disease, as assessed by different techniques, after stem cell transplantation for chronic lymphocytic leukemia. Blood. 2006;107:4563–4569. Bottcher S, Ritgen M, Dreger P. Allogeneic stem cell transplantation for chronic lymphocytic leukemia: lessons to be learned from minimal residual disease studies. Blood Rev. 2011;25:91–96. Ritgen M, Bottcher S, Stilgenbauer S, et al. Quantitative MRD monitoring identifies distinct GVL response patterns after allogeneic stem cell transplantation for chronic lymphocytic leukemia: results from the GCLLSG CLL3X trial. Leukemia. 2008;22:1377–1386. Ritgen M, Stilgenbauer S, von Neuhoff N, et al. Graftversus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR. Blood. 2004;104:2600–2602. Aalbers AM, Aarts MJ, Krol AD, et al. The beneficial local and abscopal effect of splenic irradiation in frail patients with chronic lymphocytic leukaemia. Neth J Med. 2016;74:122–129. Frinc I, Dima D, Chitic M, et al. Transthoracic ultrasonography for the follow-up of a chronic lymphocytic leukemia patient with chemotherapy-induced immunosuppression prior to allogeneic stem cell transplantation. A case report. Med Ultrasonogr. 2017;19:330–332. Frinc I, Ilies P, Zaharie F, et al. Transthoracic ultrasonography for the immunocompromised patient. A pilot project that introduces transthoracic ultrasonography for the follow-up of hematological patients in Romania. Romanian J Internal Med ¼ Revue Roumaine De Medecine Interne. 2017;55:103–116.

344

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

C. TOMULEASA ET AL.

Porter DL, Hwang WT, Frey NV, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7:303ra139. Strati P, Keating MJ, O'Brien SM, et al. Eradication of bone marrow minimal residual disease may prompt early treatment discontinuation in CLL. Blood. 2014;123:3727–3732. Dimier N, Delmar P, Ward C, et al. A model for predicting effect of treatment on progression-free survival using MRD as a surrogate endpoint in CLL. Blood. 2018;131:955–962. Sayala HA, Rawstron AC, Hillmen P. Minimal residual disease assessment in chronic lymphocytic leukaemia. Best Pract Res Clin Haematol. 2007;20:499–512. Mattarucchi E, Spinelli O, Rambaldi A, et al. Molecular monitoring of residual disease in chronic myeloid leukemia by genomic DNA compared with conventional mRNA analysis. J Mol Diagn: JMD. 2009;11: 482–487. Moravcova J, Nadvornikova S, Sieglova Z, et al. Do transcriptionally silent BCR-ABL cells persist in CML patients in molecular remission after stem cell transplantation? Leukemia. 2001;15:997–999. Sekiya Y, Xu Y, Muramatsu H, et al. Clinical utility of next-generation sequencing-based minimal residual disease in paediatric B-cell acute lymphoblastic leukaemia. Br J Haematol. 2017;176:248–257. Ladetto M, Bruggemann M, Monitillo L, et al. Nextgeneration sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299–1307. Innao V, Allegra A, Russo S, et al. Standardisation of minimal residual disease in multiple myeloma. Eur J Cancer Care (Engl). 2017;26. DOI: 10.1111/ecc.12732 Shin S, Hwang IS, Kim J, et al. Detection of immunoglobulin heavy chain gene clonality by next-generation sequencing for minimal residual disease monitoring in B-lymphoblastic leukemia. Ann Lab Med. 2017;37:331–335. Kotrova M, van der Velden VHJ, van Dongen JJM, et al. Next-generation sequencing indicates falsepositive MRD results and better predicts prognosis after SCT in patients with childhood ALL. Bone Marrow Transplant. 2017;52:962–968. Gaipa G, Cazzaniga G, Valsecchi MG, et al. Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia. Haematologica. 2012;97:1582–1593. Schrappe M. Minimal residual disease: optimal methods, timing, and clinical relevance for an individual patient. Hematol Am Soc Hematol Educ Program. 2012;2012:137–142. Hillmen P. Beyond detectable minimal residual disease in chronic lymphocytic leukemia. Semin Oncol. 2006;33:S23–S28. Robertson LE, Huh YO, Butler JJ, et al. Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic,

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

immunophenotypic, and molecular analysis. Blood. 1992;80:29–36. Vuillier F, Claisse JF, Vandenvelde C, et al. Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements. Leuk Lymphoma. 1992;7:195–204. Lenormand B, Bizet M, Fruchart C, et al. Residual disease in B-cell chronic lymphocytic leukemia patients and prognostic value. Leukemia. 1994;8:1019–1026. Cabezudo E, Matutes E, Ramrattan M, et al. Analysis of residual disease in chronic lymphocytic leukemia by flow cytometry. Leukemia. 1997;11:1909–1914. Rawstron AC, Villamor N, Ritgen M, et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 2007;21:956–964. Rawstron AC, Kennedy B, Evans PA, et al. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood. 2001;98: 29–35. Stehlikova O, Chovancova J, Tichy B, et al. Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice. Int J Lab Hematol. 2014;36:165–171. Rawstron AC, Bottcher S, Letestu R, et al. Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL. Leukemia. 2013;27: 142–149. Raponi S, Della Starza I, De Propris MS, et al. Minimal residual disease monitoring in chronic lymphocytic leukaemia patients. A comparative analysis of flow cytometry and ASO IgH RQ-PCR. Br J Haematol. 2014;166:360–368. Rawstron AC, de Tute R, Jack AS, et al. Flow cytometric protein expression profiling as a systematic approach for developing disease-specific assays: identification of a chronic lymphocytic leukaemiaspecific assay for use in rituximab-containing regimens. Leukemia. 2006;20:2102–2110. Sartor MM, Gottlieb DJ. A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2013;84:96–103. Farren TW, Giustiniani J, Fanous M, et al. Minimal residual disease detection with tumor-specific CD160 correlates with event-free survival in chronic lymphocytic leukemia. Blood Cancer J. 2015;5:e273. Dowling AK, Liptrot SD, O'Brien D, et al. Optimization and validation of an 8-color single-tube assay for the sensitive detection of minimal residual disease in bcell chronic lymphocytic leukemia detected via flow cytometry. Lab Med. 2016;47:103–111. Short NJ, Keating MJ, Wierda WG, et al. Fludarabine, cyclophosphamide, and multiple-dose rituximab as frontline therapy for chronic lymphocytic leukemia. Cancer. 2015;121:3869–3876.


[141]

[142]

[143]

[144]

[145]

[146]

[147]

[148]

Montillo M, Cafro AM, Tedeschi A, et al. Safety and efficacy of subcutaneous Campath-1H for treating residual disease in patients with chronic lymphocytic leukemia responding to fludarabine. Haematologica. 2002;87:695–700. Del Poeta G, Del Principe MI, Buccisano F, et al. Consolidation and maintenance immunotherapy with rituximab improve clinical outcome in patients with B-cell chronic lymphocytic leukemia. Cancer. 2008;112:119–128. Wendtner CM, Ritgen M, Schweighofer CD, et al. Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission–experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG). Leukemia. 2004;18: 1093–1101. Bosch F, Abrisqueta P, Villamor N, et al. Rituximab, fludarabine, cyclophosphamide, and mitoxantrone: a new, highly active chemoimmunotherapy regimen for chronic lymphocytic leukemia. J Clin Oncol. 2009;27:4578–4584. Moreton P, Kennedy B, Lucas G, et al. Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival. J Clin Oncol. 2005;23: 2971–2979. Wierda WG. Minimal residual disease provides treatment focus for next chronic lymphocytic leukemia advances. J Clin Oncol. 2016;34:3722–3723. Moreno C, Ritgen M, Rawstron A. Is MRD eradication a desirable goal in CLL? Best Pract Res Clin Haematol. 2010;23:97–107. Caballero D, Garcia-Marco JA, Martino R, et al. Allogeneic transplant with reduced intensity conditioning regimens may overcome the poor prognosis of B-cell chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene and chromosomal abnormalities (11q- and 17p-). Clin Cancer Res: Off J Am Assoc Cancer Res. 2005;11: 7757–7763.

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

345

Santacruz R, Villamor N, Aymerich M, et al. The prognostic impact of minimal residual disease in patients with chronic lymphocytic leukemia requiring first-line therapy. Haematologica. 2014;99:873–880. van Dongen JJ, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257–2317. Logan AC, Gao H, Wang C, et al. High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment. Proc Natl Acad Sci USA. 2011;108:21194–21199. Logan AC, Zhang B, Narasimhan B, et al. Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia. Leukemia. 2013;27:1659–1665. Campbell PJ, Pleasance ED, Stephens PJ, et al. Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing. Proc Natl Acad Sci USA. 2008;105:13081–13086. Sarantopoulos S, Stevenson KE, Kim HT, et al. Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease. Blood. 2011;117: 2275–2283. Varghese AM, Rawstron AC, Hillmen P. Eradicating minimal residual disease in chronic lymphocytic leukemia: should this be the goal of treatment? Curr Hematol Malig Rep. 2010;5:35–44. Nabhan C, Coutre S, Hillmen P. Minimal residual disease in chronic lymphocytic leukaemia: is it ready for primetime? Br J Haematol. 2007;136:379–392. Evans PA, Pott C, Groenen PJ, et al. Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia. 2007;21:207–214.