Early stage treatment

What follows is an early draft of a paper I am writing that makes the case for new trials of whether early treatment might benefit some patients with CLL. I would welcome comments.

Introduction

For most patients with neoplastic disease it is almost axiomatic that early diagnosis leads to earlier treatment and better outcomes. For chronic lymphocytic leukemia (CLL) there is no evidence to support this. Indeed a meta-analysis of six trials involving more than 2000 patients that compared early treatment with treatment deferred until the disease became progressive or symptomatic showed no significant difference in overall survival [1]. However, in these trials nearly half the patients in both arms were still alive after 10 years follow-up and therefore the trials must have included patients who would never under require treatment current guidelines [2]. Moreover, the patients were treated with chlorambucil, with or without prednisolone. Many authorities believe that there are now more effective treatments than that. Although there as yet no randomized clinical trials that show longer overall survivals for any first-line treatment than chlorambucil [3], the use of purine analogues alone or in combination with cyclophosphamide with or without rituximab and with or without mitoxantrone all give a higher rate of complete remission and complete remissions without minimal residual disease, as well as longer progression-free survivals [4-8].

In recent years several prognostic markers have been described that are able to predict which patients will eventually require treatment. In particular, unmutated IGVH genes [9, 10], the use of the IGVH3.21 gene [11], increased CD38 expression [9, 12], increased ZAP-70 expression [13-15] and the detection of deletions of portions of the long arm of chromosome 11 (del 11q23) or the short arm of chromosome 17 (del 17p13) by fluorescent in-situ hybridization (FISH) [16] all usefully predict poor outcome in early stage disease. Patients with del 17p13 are a group with very poor survival with disease that is resistant to most active agents [17] and because of this we have excluded them from this analysis.

In this study we have looked at the feasibility of re-examining the question of early versus deferred treatment in a new randomized controlled trial.
Methods

Patients
In this retrospective study we have examined 297 patients with Binet stage A CLL who presented at the Royal Bournemouth Hospital and have had prognostic markers performed. The following prognostic markers were studied: IGVH mutational status, use of IGVH 3.21 heavy chain gene, CD38 expression, ZAP-70 expression and the presence of deletions at 11q23 or 17p13. Those with del 17p13 were excluded from the analysis. Patients were treated according to NCI guidelines [2]. Times from presentation to first treatment were calculated and treatment-free survival times calculated for patients with nought, one, or two or more adverse factors. Overall survival curves were also calculated for the same three groups.

IGVH gene analysis
Prior to October 2004 IGVH genes were sequenced as previously described [10]. The preferred source material was RNA. cDNA was synthesized and amplified by polymerase chain reaction (PCR) using a mixture of oligonucleotide 5’ primers specific for each leader sequence of the VH1 to VH6 families or a consensus 5’ FW1 region primer, together with either a consensus 3’ primer complementary to the germ line JH regions or a 3’ primer complimentary to the constant region. From 2004 onwards gDNA was extracted from whole blood using the QIAmp®DNA mini kits (Qiagen, Crawley, West Sussex, UK) according to the manufacturers instructions. gDNA was amplified in a single multiplexed PCR reaction consisting of 6VH framework 1 primers combined with one JH consensus primer (standardises BIOMED-2 primers). Clonal sequences were determined by sequencing amplicons from at least 2 independent PCR reactions. The majority of samples were sequenced directly using an automated DNA sequencer. Nucleotide sequences were aligned to EMBL/GenBank and current databases (V-BASE sequence directory IMGT/V-QUEST, using MacVector 4.0 sequence analysis software; International Biotecnologies, New Haven, CT, and Lasegene; DNASTAR, Madison, WI.). Percentage homology was calculated by counting the number of mutations between the 5’ end of FR1 and the 3’ end of FR3. Homology with the germline sequence of 98% or more was regarded as unmutated.
CD38
CD38 expression on fresh or cryopreserved cells was determined by flow cytometry as previously described [12] using FITC labeled anti-CD5 (clone DK23; DAKO, Glostrup, Denmark), PE labeled anti-CD38 (clone HB7; Becton Dickinson, San Jose, CA) and RPE-Cy5 labeled anti-CD19 (clone HD37, DAKO). We chose a cut-off point for CD38 that give the highest possible Youden index. Cut-off points of 20% and 30% gave the highest, but similar, Youden values—58% and 60%, respectively, and in this instance we chose 20%.
ZAP-70
ZAP-70 expression on fresh or cryopreserved cells was measured by flow cytometry as previously described [14], using an indirect assay that makes use of an unlabelled anti-ZAP-70 (clone 2F3•2, Upstate Biotechnology, Milton Keynes, UK) followed by secondary antibody (Sheep-anti-mouse FITC-conjugate, Novocastra, Newcastle Upon Tyne, UK). An isotype control (mouse IgG2a, DAKO) was used to define negative staining and T and NK cells were identified using and anti-CD2-PE conjugate (DAKO). A cut-off level of 10% positivity was chosen as previously described.
FISH
Separate hybridizations were carried out for loci on chromosomes 11 and 17 as previously described [18]. LSIp53, together with CEP17 alpha satellite DNA probe labeled with Spectrum Orange and Spectrum Green (Vysis UK, London, United Kingdom), respectively, were used to evaluate chromosome deletion at 17p13.1. For chromosome 11, CEPH yacs 755b11 and 801b11 were labeled by nick translation with Spectrum Orange dUTP and Spectrum Green dUTP (Vysis), respectively, according to the manufacturer’s protocol. Hybridization was to peripheral blood lymphocytes or to cells from our archive of fixed TPA stimulated lymphocyte cultures.
Statistical methods
Data were analyzed using GraphPad Prism 4. Survival functions comparing patients have been estimated using the product limit method of Kaplan Meier.

Results

Of the 297 stage A patients there were 148 patients who had none of these adverse factors, 78 who had one of them and 71 (23.9%) who had two. Actuarial treatment-free survival curves were constructed for these three groups and are shown in the figure. Median treatment-free survival for those with no adverse prognostic factors has not been reached; for those with one adverse prognostic factor the median treatment-free survival was 123 months and for those with two it was 37 months (p<0.0001).

The median overall survival for patients with at least two adverse prognostic factors was 102 months.

Discussion

Nearly a quarter of all stage A patients presenting to a district general hospital had at least two adverse prognostic markers. Half of these required treatment according to NCI guidelines within three years of diagnosis. Given the number of patients presenting annually in the United Kingdom there should be no difficulty in accruing sufficient patients for a randomized clinical trial of early treatment versus ‘watch and wait’.

On the other hand the use of only a single adverse prognostic factor (such as unmutated IgVH genes) would mean that it would take more than ten years for half the patients to require treatment and some patients would normally remain treatment-free for more than 20 years. In view of the unproven nature of early treatment, such a trial would be ethically suspect.

Of greater difficulty is deciding which treatment should be offered. Although drug combinations that include purine analogues yield the highest complete response rates, the possibility of selecting for p53 mutant subclones has been raised [19] and risk that early intervention might generate drug-resistant disease is apparent. Furthermore, the prolonged depletion of CD4 positive T cells might be responsible for the reported higher incidence of Richter’s syndrome after fludarabine therapy [20]. To avoid the potential dangers of early chemotherapy, treatment with monoclonal antibodies might be assessed. Although single agent rituximab does produce objective responses in 51% of patients when used first line in CLL [21], complete responses are very rare.

On the other hand alemtuzumab is capable of producing complete remissions in 72% of patients without lymphadenopathy and complete remissions with the absence of minimal residual disease in 39% [22]. Although its toxicity is seen as an important drawback in multiply treated patients, when used as a first line agent its only serious toxicity is CMV viremia and this is regarded as manageable [23]. Although profound depletion of CD4 positive T cells occurs following alemtuzumab treatment, recovery is probably quicker than after treatment with fludarabine [24].

The most meaningful end-point for a trial of early treatment versus deferred treatment is overall survival. Trialists have been reluctant to use this as an endpoint for clinical trials in CLL, preferring progression-free survival as a surrogate, since patients may be expected to be long-lived and receive several rounds of subsequent therapy. However, this particular group has a median overall survival of only eight and a half years so that any difference in overall survival is likely to be apparent well before the ten year follow up of the meta-analysis quoted above.



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