Organización “A veces los compañeros se molestan con ella porque

Minimal residual disease (MRD) is the term used for small numbers of disease cells that remain during or after treatment, when the patient is in remission according to conventional criteria (Hallek et al., 2008). It has been well established that patients attaining complete remission (CR) have a better survival rate than poor responders (Wierda et al., 2005). This finding led to the concept of improving the quality of response to the greatest possible extent, up to the point of eradication of MRD. With the advent of combination

immunochemotherapy, the goal of treatment has changed from disease control in a chronic indolent disease to eradicating the disease to a point where there is no MRD detected and potentially a cure. This has increased the demand for finding newer agents, especially to treat resistant disease. Diagnosing disease at the MRD level is also challenging. It is now widely accepted that MRD negativity in CLL should be set at a threshold of less than a single CLL cell in 10,000 cells per μL, as this is the level that can be reliably detected by modern techniques (Hallek et al., 2008). This is accepted in the guidelines of the International Workshop on Chronic Lymphocytic Leukaemia (IWCLL) in 2008 (Hallek et al., 2008). Current methods for the detection of MRD in CLL use either flow cytometry or PCR. The initial flow cytometric analyses for MRD used the diagnostic technique itself, which is basically detection of co-expression of CD5 and CD19 together with monoclonality of light-chain expression. MRD was considered positive if more than 25% of CD19+ cells co-expressed CD5. Although these techniques are more

sensitive than a morphologic assessment, they are only capable of detecting a single malignant cell in about 200 normal cells. Techniques using additional antigens such as CD79b and CD20 were also described, but they were not applicable to everyone, as there are inter-patient variations in antigen expression. The PCR technique initially described used consensus primers, which amplify the immunoglobulin heavy chain (IgH) gene. Again, the

sensitivity with this technique was limited, and it was applicable to only 70% to 80% of patients because of IgH gene mutation. Later, allele-specific

oligonucleotide PCR (ASO-PCR) was developed, in which individual patient- specific oligonucleotide primers were designed to detect MRD in follow-up samples. This technique has the highest sensitivity (as low as 1 in 106) but is

expensive, labour-intensive, and impossible to perform in a significant proportion of patients, such as those whose pre-treatment sample is not available. In 2001, Rawstron et al. described a flow cytometric technique that can differentiate CLL cells from their normal counterparts on the basis of multicolour flow cytometry studying CD19/CD5/CD20/CD79b expression (Rawstron et al., 2001). This assay is rapid and sensitive, detecting one CLL cell in 104 to 105 leukocytes; it is also applicable to all patients, even when no pre-treatment specimen is available. Since then, various groups have

described other antibody combinations. In 2007, the European Research Initiative on CLL (ERIC) proposed an international standardised approach after analysing various combinations of antibodies and comparing them against the ASO-PCR technique (Rawstron et al., 2007). After analysing 728 paired blood and bone marrow samples, they derived several conclusions: 1) Blood analysis was equally or more sensitive than marrow in 92% of samples, but marrow analysis was necessary to detect MRD within 3 months of

alemtuzumab therapy; 2) The κ/λ/CD19/CD5 combination can be used to screen samples and avoid extended analysis in cases with clear evidence of residual disease where all B-cells are CD5+ with light-chain restriction; 3) A CD45/CD14/CD19/CD3 combination or an equivalent can be used to provide a control for CLL cell enumeration and to define the limit of detection; 4) The combination of CD5/CD19 with CD20/CD38, CD81/CD22, and CD79b/CD43 is the best panel to detect MRD with low inter-laboratory variation, low false detection rates and an accuracy of 95.7%. Current methods involve either using allele-specific PCR or flow cytometry. The sensitivity of both techniques is similar, but the PCR technique has several practical limitations. The current flow cytometric technique uses a combination of several antibodies for an accurate estimation of the minimal residual disease. Even though a

combination of CD5/CD19 with CD20/CD38, CD81/CD22, and CD79b/CD43 is the best panel to detect MRD, the search for an ideal antibody or

combination of antibodies is still continuing.

Over the years several studies have looked into the difference in survival between patients who attained MRD negativity and those who have not after their standard treatment. Most of these studies have concluded that patients who attained MRD negativity will have longer response duration and some of them have shown survival advantage (O’Brien et al., 2003) (Bosch et al., 2002) (Del Poeta et al., 2005) (Tam et al., 2008) (Hillmen et al., 2007) (O’Brien et al., 2003). Attainment of MRD negativity has been demonstrated as an independent predictor of OS and PFS by Kwok et al (Kwok et al., 2009).

In this study, data was collected retrospectively from 137 patients who attained at least a PR after their standard treatment, and in whom an MRD assessment was done using a sensitive four-colour flow cytometry.

Multivariate analysis showed that achieving MRD negativity in CLL is an independent predictor of survival even with a variety of different treatment approaches and regardless of the lines of therapy. In patients after their first line of treatment, the 5-year PFS was 89% (95%-CI 55-97%) vs. 0% (95%-CI <1%) (p<0.001) and the 5-year OS was 95% (95%-CI 61-99%) vs. 53% (95% CI-15-74%) (p<0.001) for MRD-negative vs. MRD-positive patients

respectively. This data suggests that achieving MRD-negativity after first-line therapy has a profound effect on survival. The most convincing evidence is from the German CLL8 trial which was a randomised control trial assessing the efficacy of FC vs. FCR in previously untreated patients (Hallek et al., 2010b). MRD levels were prospectively quantified in 1,775 blood and bone marrow samples from 493 patients from both arms. Patients were categorised into different MRD groups according to the level of persistent disease- low <10-4, intermediate ≥ 10-4 to <10-2, and high ≥10-2. Median PFS was 68.7, 40.5, and 15.4 months for low, intermediate, and high MRD levels,

respectively and median OS was 48.4 months in patients with high MRD and was not reached for lower MRD levelswhen assessed 2 months after therapy. When compared with patients with low MRD level there is a greater risk of disease progression with intermediate and high MRD levels (hazard ratios, 2.49 and 14.7, respectively; both P < .0001). In multivariate analyses that included the most important pre-therapeutic risk markers in CLL, MRD remained an independent predictor for OS and PFS. Another important observation is that PFS and OS did not differ between FC and FCR arms once MRD is attained, even though FCR has higher tendency to induce low MRD levels more frequently than FC (Böttcher et al., 2012). Several small trials have looked into consolidation treatment after standard chemotherapy. The only randomised control trial trying to address it prospectively was prematurely stopped due to toxicity issues (Wendtner et al., 2004). But long- term follow up of the small cohort of patients who were consolidated with alemtuzumab has shown that there is a significant survival advantage for the patients who attained MRD negativity post-consolidation (Schweighofer et al., 2009). A recent UK trial, CLL207, was a phase 2 trial which assessed

alemtuzumab consolidation post-chemotherapy in patients who responded with low levels of disease. MRD eradication from blood and bone marrow was

attained in 83% of patients at the end of alemtuzumab consolidation. The long-term survival data from this trial is still awaited (Varghese et al., 2010a).