Friday, October 19, 2007

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.


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.

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 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 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.
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.


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.


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.

1. CLL Trialists’ Collaborative Group. Chemotherapeutic options in chronic lymphocytic leukaemia: a meta-analysis of the randomised trials. J Natl Cancer Inst 1999;91:861-8.
2. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S et al. National Cancer Institute-Sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:4990-7.
3. Steurer M, Pall G, Richards S, Schwarzer G, Bohlius J, Greil R; Cochrane Haematologic Malignancies Group. Single-agent purine analogues for the treatment of chronic lymphocytic leukaemia: a systematic review and meta-analysis. Cancer Treat Rev. 2006;32:377-89.
4. Rai KR, Peterson BL, Appelbaum FR, Kolitz J, Elias L, Shepherd L et al. Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. N Engl J Med 2000;343:1750-1757.
5. O'Brien SM, Kantarjian HM, Cortes J, Beran M, Koller CA, Giles FJ et al. Results of the fludarabine and cyclophosphamide combination regimen in chronic lymphocytic leukemia. J Clin Oncol. 2001;19:1414-20.
6. Bosch F, Ferrer A, López-Guillermo A, Giné E, Bellosillo B, Villamor N, et al. Fludarabine, cyclophosphamide and mitoxantrone in the treatment of resistant or relapsed chronic lymphocytic leukaemia. Br J Haematol. 2002;119:976-84.
7. Keating MJ, O’Brien S, Albitar M, Lerner S, Plunkett W, Giles F et al. Early results of a chemoimmunotherapy regimen of fludarabine, cyclophosphamide, and rituximab as initial therapy for chronic lymphocytic leukemia J Clin Oncol 2005;23:4079-88.
8. Wierda W, O’Brien S, Faderl S, Ferrajoli A, Wang X, Do K-A et al. A Retrospective comparison of three sequential groups of patients with recurrent/refractory chronic lymphocytic leukemia treated with fludarabine-based regimens. Cancer 2006;106:337–45.
9. Damle RN, Wasil T, Fais F Ghiotto F, Valetto A, Allen SL et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 1840-7.
10. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848-54.
11. Tobin G, Thunberg U, Johnson A, Thorn I, Soderberg O, Hultdin M. Somatically mutated Ig VH3-21 genes characterize a new subset of chronic lymphocytic leukemia Blood. 2002;99:2262-4.
12. Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002, 99: 1023-1029.
13. Crespo M, Bosch F, Villamor N Bellosillo B, Colomer D, Rozman M et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med. 2003;348:1764-1775
14. Orchard JA, Ibbotson RE, Davis Z Wiestner A, Rosenwald A, Thomas PW et al. ZAP-70 expression by flow cytometry is a good prognostic marker in CLL and a potential surrogate for immunoglobulin VH gene mutations. Lancet 2004 363:105-111.
15. Rassenti LZ, Huynh L, Toy TL Chen L, Keating MJ, Gribben JG et al. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004 351:893-901.
16. Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L et al Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910-6.
17. Catovsky D, Richards S, Matutes E, Oscier D, Dyer MJS, Bezares RF et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet 2007; 370:230–9
18. Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors Blood.2002;100:1177-84
19. Rosenwald A, Chuang EY, Davis RE, Wiestner A, Alizadeh AA, Arthur DC et al. Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. Blood. 2004;104:1428-34.
20. Thornton PD, Bellas C, Santon A, Shah G, Pocock C, Wotherspoon AC et al. Richter's transformation of chronic lymphocytic leukemia. The possible role of fludarabine and the Epstein-Barr virus in its pathogenesis.
Leuk Res. 2005;29:389-95.
21. Hainsworth JD, Litchy S, Barton JH, Houston GA, Hermann RC, Bradof JE et al. Single-agent rituximab as first-line and maintenance treatment for patients with chronic lymphocytic leukemia or small lymphocytic lymphoma: A phase II trial of the Minnie Pearl Cancer Research Network . J Clin Oncol 21:1746-51.
22. Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood. 2002;99:3554-61.
23. Hillmen P, Skotnicki A, Robak T, Jaksic B, Sirard C, Mayer J. Alemtuzumab (CAMPATH ®, MABCAMPATH®) has superior progression free survival (PFS) vs chlorambucil as front line therapy for patients with progressive B-cell chronic lymphocytic leukemia. Blood 2006;108:93a (abstract 301)
24. Lundin J, Porwit-MacDonald A, Rossmann ED, Karlsson C, Edman P, Rezvany MR et al. Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia. Leukemia 2004;18: 484–90.


Anonymous said...

Clearly for young patients with so-called "high-risk" CLL such studies are important, Studies going forward clearly should include patients with 17- and may need to be designed so the results can be compared statistically to accepted regimens such as FR orFCR or HDMP+C,

The other question that needs to be addressed is the utuility of such therapy as a prelude to SCT or other potentially curative therapies (ie, vaccines or TCell engineering) that may becime avaialable in the future.

As A 56 yo man with "high-risk" CLL and an ever increasing ALC I find the lack of data appalling.


dave said...

Thanks for the post, it was very informative. I was curious, in the patients with only one unfavorable marker, how many of these had unmutated as their only poor indicator? The question has special interest to me since I have no poor prognostic factors except that I am only 1.7% mutated. Thanks for any info.

Jenny Lou said...

This is the information that I am following like a watch dog. Early intervention for the "aggresive" CLL. Tom will be one of the cller's in future data. He was dx in Dec. 2003 and started FCR3 in April 2004. He hit a nodular PR, slowly backing into a PR and now is relapsing with swollen liver and 3 lymph areas swollen, sweats, fatigue and a possible reactivation of either mumps or EBC virus. He is Zap70+ at 40%, done at UCSD, unmutated at 98.4% and although at dx his CD38 was 1.4%, it is now in the high 20's. It has climbed higher with every flow since FCR. Next treatment in Feb/March or as soon as it is released will be Revlimid plus Rituxan. Interesting side is that his lymphocyte count is still fairly low---the issue seems to keep coming back to SLL.
For your study, I would suggest Campath to be the drug involved.

Jenny Lou said...

Second thought on this "early stage treatment". I really have a hard time understanding giving a protocol for CLL that has been proven not to be long lasting. I understand that at this point we can only do palliative care. So--the,"cure", if there ever is one, will come from a trial. So--I believe that early stage treatment for patient's with aggressive marker's is the smartest approach. We already know that there is a low life expectancy with this group. I remember seeing a chart that you did Terry, on unmutated patients that had a straight line to zero at 14 years from dx. Is that correct? So, you see, when one is dx at the age of 49--should we wait like Kurt did? I don't believe so. We know the outcome of watching and waiting. The jury is still out on early stage treatment and therein lies the advantage. What I am amazed about is that every single researcher/Dr. is not jumping on this immediately. Please don't debate the precious time we have left to death. That's my view and I'm sticking by it. Thanks Terry, for posting this.

Anonymous said...

First things first...'Nought'? Hmmm, how about 'none' or 'zero'? Nought is such an 'English' word that my spellcheck program marks it as a misspelling.

On to your study.

How can you control for subsequent treatment? Even if all of these patients remain with your trial site, differences certainly will remain as to when a patient needs subsequent treatment. I submit that it would be unethical not to allow your study patients freedom in selecting diverse treatment options.

However, since there are few such options in the state-run health plan (NHS), maybe that isn't a big problem in England. It would be in the US. Different subsequent treatments would mean that your statistics using over-all survival would be meaningless. I'd suggest treatment-free survival as a better end point. That would also be more ethical in my book.

Unlike most of you all, I have less of a problem with historical controls. Variables tend to even out as the sample grows larger and larger. Take MD Anderson's use of FCR, which I'm sure they consider the 'gold standard'. Given the numbers of patients they have treated with this regime (and given the length of time it has been used), it seems obvious that if non-subtle improvements in survival are apparent, that is a strong argument that FCR is superior to other treatments in use even today.

The use of single agent Campath is somewhat ethically unjustifiable, given that FCR does give superior complete remissions and over-all response rates than does single agent Campath. But perhaps FCR isn't offered by the NHS. Too expensive.

I suppose if you find enough patients who are willing to live by your rules, the results might be quite interesting. You could authoritatively state whether early Campath did improve survival.

It's certainly a long-term project, so there is job security as well.

Would I sign up for it? Very possibly yes, if I had the freedom to decide myself what my second-line treatment would be.

Terry Hamblin said...

Dave your answer is 23.

Jenny-Lou. The only drawback with Campath is its failure to penetrate large lymph node masses, so that would be a contraindication.

Anonymous. Thanks for the information on 'nought' I'm surprised that it is not understood by Americans. It is certainly the most commonly used word for zero in the UK.

Of course we cannot control subsequnt treatment. I assume that whether treated early or observed, the next treatment will be the best availavble. Progression-free survival would not be an adequate test because it would hardly be a surprised that untreated patients would show progresion first. The point about early treatment is to make patients live longer. If they don't then there is no point in doing it.

I am not convinced by the MD Anderson data on FCR because a far higher proportion of patients receiving FCR were stage I and II compared with their own series receiving FC. Apparently better results might be due to less ill patients. Next year we should have some definitive head-to-head results. If FCR shows an improvement in PFS over FC it will become available on the NHS.

Anonymous said...

I can go with a study of 2 'bad' markers novel treatment using historical data as the 'other' arm of the trial, but I can't see the benefit to starting treatment before the conventional intiation point. anything that extends that 'no treatment' time is a bonus, since treatment has other detrimental effects on health. JHB.

Richard said...

You say treating early or late makes no difference to overall patient survival.

Now treatment takes a toll on normal as well as cancerous cells and leaves patients open to infections (viral and bacterial). It can also trigger some transitions to AIHA and other more serious complications and possibly death.

Since there is no difference in overall survival between early and late treatment does this mean that late treatment is somewhat less effective than early treatment, to keep the books balanced?

Have there been any studies to see if length of remission after treatment is correlated with doubling time measured before treatment?

Terry Hamblin said...

Early treatment carries the risk of treating people who will never need treatment otherwise. These patients will do worse than those who never get treatment. On the other hand there are some patients who will benefit from early treatment, and these two will balance out. However, I think these early trials are of little value now, because we have new drugs and better ways of predicting what will happen to individual patients.

There are no studies such as you suggest.