Thursday, January 13, 2011

Stereotypy and CLL

Although some are obsessed with finding surrogates, I still support the test for IGHV mutations as one of the best prognostic markers for CLL. CD38, ZAP-70 and CD 49d all have their advocates, but each has its drawbacks and the only thing in favor of them is that most labs now have a flow cytometer. However, a gene sequencer is no more expensive to buy and the sequencing of IGHV genes has became a largely automated test. I saw a paper the other day which had collected gene sequences from over 8000 CLL patients.

There have been developments in IGHV testing that have made it a much more interesting tool. They can be summed up by the word 'stereotypy'.

When I first stared talking to Nick Chiorazzi about IGHV genes he told me about five sequences he had determined from patients who originated from all over the world. They had all used the same IGHV gene, V4-39, and they all had surface IgG rather than IgM+D. Furthermore they all had the same Ig sequence. Since there are a possible thousand billion different sequences, it was difficult to believe that there hadn't been some cross contamination. But Nick assured me that they had checked for this and the identity was confirmed.

Then Gerrard Tobin and Richard Rosenquist produced the astonishing finding that patients whose CLL used the V3-21 gene had a poor prognosis whether they were mutated or unmutated. When I saw the sequences it became clear that many of the V3-21 had identical or near-identical sequences. It all suggested that at least for some CLLs the leukemia had originated in a clone of B lymphocytes that was expanding in response to stimulation by a single antigen.

The question then was, if there are these two families of cases with identical sequences, are there others? Enter Kostas Stamatopoulos from Greece. In collaboration with workers in many countries he has established that approximately one patient in three has a IGHV sequence that belongs to a stereotyped family. Moreover for some of these there is a clinical correlation - all members of the family behave similarly, and the originating antigen has been determined. For some of the families the members are too few for us to be accurate about how they will behave, which provides a justification for everybody to have their V genes sequenced. Sequences that were near-identical for a number of patients were given the name 'stereotypes'.

Here are some examples:
Stereotype #2 V3-21 mutated or unmutated, poor prognosis, antigentic stimulation by coffin-1, high frequency del 11q23.
Stereotype #8 V4-39, unmutated, surface IgG, poor prognosis, antigenic stimulation by vimentin, 17 fold risk of Richter's transformation, high frequency of trisomy 12.
Stereotype #4 V4-34, mutated, good prognosis, presentation at young age (~43), antigenic stimulation by Ii blood group.
Stereotype #16 V4-34, mutated, good prognosis - though not as good as #4.
Stereotype #1 V1-5-7, unmutated, poor prognosis, antigenic stimulation by vimentin.
Stereotype #5 V1-69, umutated, antigenic stimulation by coffin-1, favorable prognosis.
Stereotype #6 V1-69, unmutated, antigenic stimulation by non-muscle myosin heavy chain, prognosis intermediate.

It can be seen that family identity over-rides both the particular V gene used and the mutational status. There is obviously a lot more to come from this approach.


Anonymous said...

Fascinating - and begins to make some sense of what currently appears to be a very random course of disease progression when looking at individual patients who have similar markers but who have very different rates of disease progression.

This suggests to me that there is some logic in Cll disease progression - we just have not understood what the relationship was. v

Anonymous said...

Is this "tool" now being used or is it still too early?

Would any of these prognostic indicators change how you might approach a patient's treatment- both type of treatment and timing of treatment?

It is amazing how many "things/information" seem to be "in play" these days in the CLL world.

One can only hope they can be translated into better results for patients....

Thanks for the, as always, cogent analysis.

Terry Hamblin said...

Still early days yet.

Anonymous said...

Could you please explain better the difference between how the particular V genes utilized are identified & how, exactly, they are then identified a mutated or not. In my case the Mayo Clinic advised me that I had 100% homology (unmated), but never identified whether any particular V genes were used.

Thank you

Terry Hamblin said...

It is a matter of extracting the DNA from the CLL cells and by using the appropriate primers then PCR you can expand the particular region of the DNA that codes for the V-gene and then sequence this are using a gene sequencer. The 51 VH genes are known so one then compares the sequence of the patient's DNA from the known germ line sequence and count how many changes there are. This is then expressed as a percentage.

Anonymous said...

So does this mean some people can be classified as unmutated, but still have a good prognosis?

Terry Hamblin said...

A relatively good prognosis. this was always the case, we just had difficulty picking them.

Wayne said...

This is most intriguing and begs the question of the direction of research based on new and cheaper scanning technologies.

With the formal separation of MBL from CLL/SLL would it make sense to periodically scan patients diagnosed with MBL using appropriate scanning technologies to gain an understanding of pathogenesis and progression charting of disease based on chromosomal damage?

Could you consider blogging on the utility and differences of the various scanning techniques? I have read about Ditagging, HD Oligonucleotide and ROMA scanning but do not understand enough to know their strengths and weaknesses are.

Does this "stereotypy" perspective add to or change the predictability of clonal evolution? I sense that this new way of subtyping, since it is associated with specific factors of antigen stimulation, is moving closer to causation which should translate to earlier and more specific interventions. Very Exciting development!

Thanks much for this and the best of luck to you for what you must endure. We are all thinking of you Terry.


Terry Hamblin said...

"Ditagging, HD Oligonucleotide and ROMA" I may get to these. I am currently reading the ASH abstracts so I am a long way behind. I was extremely fatigued in Decenber and early January and I think my line infection had been going on for several weeks.

Anonymous said...

Is it possible to have unmutated gene using VH4-34 gene segment and 11q del as defined by FISH and still have a good prognosis for disease progression?

Terry Hamblin said...

Anything is possible but I think it unlikely. Unmutated 4-34s are unusual and we would need to look at a large population of these to assess likely performance.

Anonymous said...

What about sterotypy #4-59? Hope this post finds you improving.

Terry Hamblin said...

About 15% of those using V4-59 are stereotyped. I don't have any further information yet about this subset.

DKW said...

Although not quite as high-tech as this stereotypy stuff, could we sometime have your current thoughts on using R-FC in CLL patients with Coombs positivity.Does the risk ,if there is one, of aggravating the haemolysis have anything to do with the mutational status?
If (R)FC is not to be used, what apart from steroids, is safe in Coombs positive patients? I am aware of the CLL4 stuff supporting use of FC in these patients, but is it really safe?
Since the Coombs positivity often persists despite a Hb response to steroids, when does it become 'safe' to introduce a purine analogue?
I look forward to your comments on this with inteest

Terry Hamblin said...

AIHA is much commoner in unmutated cases. FCR or FC seems to reduce the risk of reactivating AIHA but the risk is not totally abolished. C and R are both used to treat AIHA and this is perhaps why they ameliorate the effect of F. Personally I like patients to be on cyclosprine before risking adding further F even with C and R.

Jacob Gunn Glanville said...

What was the publication you mentioned? The one that released 8000 CLL sequences?

Terry Hamblin said...

It was an abstract at ASH in 2010. It did not list the sequences, merely that nearly 8000 had been done.

Anonymous said...

Dear Terry,

Do you know anything about prognostic significance of IgVH mutated gene VH3-11?

Your response is much appreciated,

Howard Paul

Terry Hamblin said...

Howard in its unmutated form V3-11 is associated with Richter's transformation and PLL, but not in the mutated form with which I can find no associations.

Anonymous said...

Thank you very much Terry.


Anonymous said...

Dear Dr. Hamblin - my thoughts and prayers are with you. Bless you for all you do for the CLL community.

I have a question about mutated IgVH. Is it possible to have multiple IgVH mutations? My IgVH is 4.5% IgVH mutated (VH 3-7 variety). Could someone with a mutated IgVH (VH 3-7) also have the unfavorable VH 3-21 mutation? If yes, then IgVH would need to also test for the unfavorable VH 3-21. Can you clarify -

Thank you - Patti K

Terry Hamblin said...


Although rarely patients can have two separate clones this is very unusual and one would expect it to be picked up at the same time as the first sequence.