CLL diagnosis and prognosis: implications of the IWCLL guidelines
Roger Owen, Haematological Malignancy Diagnostic Service (HMDS)
Roger Owen, Haematological Malignancy Diagnostic Service (HMDS) Laboratory, Department of Haematology, St James’ Institute of Oncology, Leeds

Introduction
The new guidelines from the International Workshop on Chronic Lymphocytic Leukaemia (IWCLL)¹ have important implications for diagnosis, identification of prognostic factors and assessment of the patient’s response to treatment, and demonstrate the increasing importance of the haematopathology laboratory in the management of individual patients with chronic lymphocytic leukemia (CLL).¹ They emphasise the key role played by a range of investigations, such as immunophenotyping and fluorescence in situ hybridisation (FISH) analysis.¹

CLL diagnosis
The formal criteria for the diagnosis of CLL in the peripheral blood require the presence of at least 5x10⁹/L monoclonal B cells with an appropriate immunophenotype.^1,2 The guidelines consider that immunophenotyping (typically by flow cytometry) is essential for diagnosis, even if the clinical and morphological features are highly suggestive of CLL.¹ Typically, CLL cells co-express CD19, CD5 and CD23 in the presence of weak expression of CD20, CD79b and surface immunoglobulin (Ig).² This immunophenotype allows CLL to be distinguished from other lymphoproliferative disorders, such as mantle cell lymphoma and marginal zone lymphoma.¹ Weak or absent expression of CD23, particularly if there is stronger expression of CD20, CD79b or surface Ig, should raise the possibility of these alternative diagnoses, and trigger additional investigations, such as FISH.^1,2 Other antigens that are differentially expressed in CLL when compared with normal B cells or other lymphoproliferative disorders include CD40, CD81, CD22, CD21 and LAIR-1.³

The 5x10⁹/L threshold for B cells was introduced in recognition of the fact that large numbers of asymptomatic individuals (approximately 5% of those aged >60 years) have low levels of circulating clonal B cells with a CLL immunophenotype.⁴ A recent publication from our laboratory has demonstrated that these cells have the immunophenotypic and genotypic features of favourable risk CLL, and that the risk of progression to symptomatic disease requiring therapy is low at approximately 1% per annum.⁵ This clinical scenario is very reminiscent of that seen in monoclonal gammopathy of undetermined significance (MGUS). It is in recognition of these clinical outcome data that patients with less than 5x109/L circulating B cells in the absence of cytopenia and nodal disease should be diagnosed with monoclonal B-cell lymphocytosis rather than CLL.²


The usefulness of immunophenotypic studies goes beyond confirmation of the diagnosis. These investigations also demonstrate whether cells from individual patients are suitable for monitoring of minimal residual disease, and are important in the identification of new antigenic targets for monoclonal antibodies, such as CD52, CD23 and CD80.

Significance of prognostic factors
Biological prognostic factors are becomingly increasingly relevant to the management of patients with haematological malignancy, particularly in CLL, where they are essential for the evaluation of clinical trial data and are emerging as important in the management of individual patients. However, it is essential to note that such factors should be considered complementary to, rather than a replacement for, conventional staging. Moreover, prognostic factor analysis should be used to identify patients who are likely to have a good outcome as well as those expected to have an adverse outcome.

Cellular prognostic factors for CLL include:

• Cytogenetic abnormalities, i.e. presence or absence of the genetic deletions 13q14, 17p13 and 11q22 (del13q14, del17p13 and del11q22)
• Mutational status of immunoglobulin heavy chain variable (IGVH) region genes

FISH studies
Conventional metaphase cytogenetic analysis is of little or no value in CLL because of the low proliferation rate of the clonal B cells. FISH utilising interphase cells is therefore the method of choice for demonstrating the key genetic abnormalities in CLL.¹ Interphase FISH uses fluorescently labelled probes specific for the genetic loci of interest (see below); the abnormalities are detected by assessing the number of hybridisation signals present within individual cells.

The IWCLL suggests that FISH studies should be performed:¹

• In all clinical trials
• For all patients before starting treatment
• Before all subsequent therapies—this is particularly relevant for patients with refractory disease or remission of short duration

Abnormalities detected by FISH in CLL

In 2000, Döhner and colleagues used FISH analysis to demonstrate del13q14, del11q22 and del17p13 in patients with CLL.⁶ Del13q14, when present as a sole abnormality, appeared to define patients with favourable-risk CLL, in terms of median time to treatment and median overall survival (OS), but del11q22 (which results in loss of the ATM locus) and del17p13 (loss of p53) were associated with significantly adverse outcomes (Table 1). Trisomy 12 is also common in CLL (about 16% of patients), but it seems to have a relatively neutral effect on outcome.

Table 1: Effect of genetic deletions on outcomes in CLL⁶

Cytogenic abnormality (detected by FISH)	Proportion of patients	Median time to treatment (months)	Median OS (months)
Del13q14	55%	92	133
Del11q22	18%	13	79
Del17p13	7%	9	32

Translocations involving the immunoglobulin heavy chain locus at 14q32 appear to be rare events in CLL. The only consistently reported translocation appears to be the t(14;19)(q32;q13), which deregulates BCL3 as a consequence of its juxtaposition with immunoglobulin heavy chain locus. This translocation appears to be associated with atypical phenotypic features and possibly a poor clinical outcome.⁷

The overall incidence of the key cytogenetic abnormalities listed in Table 1 will vary from laboratory to laboratory because of differences in clinical practice and laboratory protocols. However, their prognostic significance has been confirmed by many studies, including the CLL4 trial in the UK.⁸

FISH in practice
Interphase FISH is readily performed on peripheral blood smears as long as there is a significant lymphocytosis. Once a laboratory is equipped to perform FISH, the assay is relatively simple and inexpensive. However, the start-up costs are considerable; laboratories need to acquire expensive hardware, such as fluorescence microscopes and specialised hybridisation equipment.
 
Various commercial kits are available. The most widely used is a two-component assay developed by Abbott Molecular Diagnostics (AMD).  In the first component, a centromeric probe for chromosome 12, labelled with a green fluorochrome, is used in conjunction with probes specific for 13q14 (red label) and 13q34 (blue label). A normal cell containing two copies of chromosome 12 and 13q will give two green, two red and two blue signals, whereas a cell with trisomy 12 will have an additional green signal, and a cell with 13q14 deletion will demonstrate a single red signal.
These hybridisation patterns are shown in Figure 1.


Figure 1: FISH analysis for trisomy 12 and del13q14

The second component of the AMD assay uses probes specific for 17p13 and 11q22, labelled with red and green fluorochromes, respectively. Again, a normal cell will contain two red and two green signals, whereas cells with del17p13 will lose one red signal, and those with del11q22 will lose one green signal. These hybridisation patterns are shown in Figure 2.

Figure 2: FISH analysis for del17p13 and del11q22

IGVH sequence analysis
IGVH sequencing has been widely used in the investigation of B-cell disorders. It provides powerful evidence of the maturational status of the underlying B-cell clone, and may give useful insights into the pathogenesis of some disorders. However, it is only in CLL where this technique has demonstrated value as a prognostic factor.

The findings of two studies published in the same issue of Blood ^9,10 show that patients with CLL characterised by germline IGVH genes have a poorer outcome than those with mutated IGVH genes, and that the effect is independent of clinical stage. These results have been corroborated subsequently by a large number of other studies. Typically, about 50% of patients have germline IGVH genes and 50% have mutations.^9,10

IGVH sequencing is now a relatively straightforward technique, with optimised strategies for generating clonal IGH rearrangements and technologies for high-throughput sequencing. Although there is much debate regarding the optimal definition of germline and mutated sequences, current convention uses =98% homology to define germline IGVH genes.¹¹
 
The IWCLL guidelines recommend assessment of IGVH status in clinical trials, but do not recommend its application in routine clinical practice. There may however be some merit in assessing newly diagnosed patients with stage A disease (see below).

Clinical utility of prognostic factors
It is important to recognise that the value of the cellular prognostic markers detailed above is very much determined by the clinical context in which they are demonstrated.
 
At present the most important application of FISH analysis is to identify those patients who have 17p13 deletions, which comprise approximately 5–10% of patients with symptomatic CLL.⁶ These patients are typically refractory to both alkylating agents and purine analogues, which is not surprising given the role of p53 in mediating apoptosis in cells exposed to DNA damaging agents. The adverse effect of 17p13 deletions is demonstrable only in those patients with a high level (>20%) of deletions.⁸  There is a clear rationale therefore for treating such patients with agents that kill CLL cells through p53-independent means, such as monoclonal antibodies and corticosteroids. It seems likely that alemtuzumab-based therapy is most appropriate in this settings and that allogeneic transplantation should be considered in responding patients.^12–16 It is important to recognise that del17p13 can emerge over time, so repeat analysis is warranted before each cycle of therapy.¹

Del11q22 is also associated with an adverse outcome, but there is not yet any clear evidence for alternative treatment strategies for CLL in such patients.

Although the IWCLL guidelines only advocate FISH in patients prior to therapy there is some merit, in my view, in performing the analysis in stage A patients. In this context, patients with deletion 13q14 as a sole abnormality would be predicted to have a favourable outcome.⁶ Similarly if IGVH analysis is to be performed in the routine setting then it is most applicable to stage A disease, since patients with mutated genes could also expect a favourable outcome.^9,10 This is powerful information to convey to a patient faced with the worry and uncertainty of newly diagnosed CLL. Adverse prognostic features, such as 17p13 and 11q22 deletions, are of course demonstrable in stage A patients, but there is no evidence as yet to suggest that early intervention improves overall outcome. IGVH analysis has less prognostic relevance in patients with symptomatic disease requiring therapy. 

Assessment of response to treatment
Assessment of so-called minimal residual disease (MRD) is becoming increasingly relevant in CLL as the efficacy of treatments improve. It is possible to demonstrate very low levels of disease using either flow cytometry or quantitative polymerase chain reaction (PCR)-based methods.^17–19 Flow cytometry is generally regarded as the method of choice because more than 95% of patients have an informative phenotype, and the technique is ideally suited to routine practice, where real time results are required for clinical decision making.¹⁸ Flow cytometry also has a reproducible sensitivity of detection of 0.01% and international standardised approaches have been developed.¹⁸

Assessment of MRD by multiparameter flow cytometry is possible because of the phenotypic differences seen between CLL cells and normal bone marrow and peripheral blood B cells.³ The assay can be performed in the peripheral blood although marrow assessments are required during monoclonal antibody therapy and for approximately 3 months following its completion. Achieving MRD negativity appears to be a very powerful predictor of outcome, irrespective of the treatment used.²⁰ This is recognised in the IWCLL guidelines, which recommend MRD assessment in future clinical trials.1 MRD assessment by flow cytometry also has a role in the assessment of patients following allogeneic transplant procedures.¹⁹  

Conclusion
The IWCLL guidelines emphasise the critical role of laboratory assessments in the management of patients with CLL. The way in which such services are delivered varies across the UK at present. There is, however, a clear rationale for the key technologies and expertise to be provided by specialist laboratories serving one or more cancer network.  

References

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