VH status

There are a number of articles authored by Gerrard Tobin and Richard
Rosenquist from Sweden that have looked at the use of VH 3-21 in CLL. They find  that
such patients have the same prognosis whether they are mutated or  unmutated.
However almost all of these have 3-4% mutations and few oif any have  more
than 7% mutations. Also the cases that they refer to mostly use the same  light
chain and the same D and J segment genes. There is some evidence that  those
patients who use VH 3-21 who don't use this light chain gene and these D  and J
segments, don't obey the Swedish rule. This was the finding of workers in
France, Spain and Italy. It may be that there is an ethnic difference between
Northern and Southern Europeans.

I promised that I would comment on apparent discrepencies in the VH mutation test when done in different labs. How the test is done: For those who want a technical description see the small print at the bottom of this letter, but here is a summary As originally done it involved extracting RNA from the leukemia cells and amplifying it with PCR using appropriate primers to get the reation going. In lymphoma you have to compare the RNA from several cells, since there are minor variations between cells (caused by ongoing mutations) and many contaminating normal B cells. The contaminating B cells can also be a problem in low count CLL, but since there is no ongoing mutation in CLL all the tumor cells should be identical. (This is not precisely true, but for practical purposes it is. Occasionally you can pick out a cell that has an extra mutation, but it does not seem to go on and form a sub-clone the way it does in follicular lymphoma). Once amplified the RNA was sequenced and its sequence compared with the 51 possible VH genes available for perusal on the databases from the internet. There are at least 2 databases in current use and they do differ slightly from each other Since the method was published in 1999 we have refined it and we now use DNA which is much more robust and travels better. The problem with DNA is that there are two #14 chromosomes that carry the VH genes and both may be active. It is therefore possible (though unlikely) that the wrong one may be amplified. If we get two bands and both are possible, the we have to revert to RNA. Sometimes people with CLL actually have two unrelated clones of CLL (after all it is a common disease so why shouldn't you get it twice?). So discrepencies are possible with VH gene assays. There could be two clones. There could be both chromosomes active. A cell with an extra mutation in it could be amplified by chance. A different database might be used for comparison. generally when discrepencies occur it is in cases with mutated VH genes, so in terms of prognosis it makes no difference. Then there could be that bugbear of all laboratories, transcriptional error. Putting the wrong name on the bottle. So far in our lab we have not found any discrepencies when we have resequenced samples, but then all our tests are done by an experienced scientist who helped to develop the test and is well aware of the pitfalls.We do the whole testing in duplicate and have always got the same result in both assays. Terry Hamblin Preparation of cDNA and DNA. Blood samples for testing were taken during the past 5 years. Some were tested immediately, while for others, the lymphocytes were cryopreserved and tested later. Because the VH gene signature is believed not to change during the clinical course of CLL, it was deemed satisfactory to determine this at any stage of the disease whether treated or not. The preferred source material was RNA, as this reduces the possibility of amplifying an aberrantly rearranged VH gene; cDNA was synthesized by reverse transcription using an oligo(dT) primer as previously described. Where RNA was not available, genomic DNA was extracted using the QIAmp blood kit (Qiagen, Hilden, Germany). Amplification of VH genes. One fifth to one third of a sample of cDNA was amplified by polymerase chain reaction (PCR) using a mixture of oligonucleotide 5' primers specific for each leader sequence of the VH1 to VH6 families, together with either mixed 3' primers complementary to the germ line JH regions or 3' primers complementary to the constant region. When there was failure to amplify, an alternative mixture of 5' primers specific for framework 1 region of VH1 to VH6 was substituted. In our hands, the VH1 leader primer also amplifies sequences from the closely related VH7 family. In all cases, PCR was performed in a final volume of 50 µL with 20 pmol of each primer, 50 µmol deoxyribonucleotide triphosphates (dNTPs), and 2.5 U Taq DNA polymerase with reaction buffer (Boehringer, Lewes, E Sussex, UK). Amplification consisted of an initial denaturation step of 3 minutes at 94°C followed by 30 cycles of 94°C, 56°C and 72°C for 45 seconds each, with a final extension step of 10 minutes at 72°C. All PCR reactions were performed in duplicate. For each PCR, a control with no added template was used to check for contamination. Sequencing and cloning of PCR products. 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 (Applied Biosystems, Foster City, CA). However, for the first 35 cases, cloning of gel-purified products into pGEM-TA vector was performed. After transformation of JM109 competent cells, clones found to contain an insert of appropriate size by restriction analysis of plasmid DNA were sequenced. A minimum of 5 bacterial colonies were analyzed. In addition, in 3 later cases, direct sequencing was unsuccessful and the sequence was determined by cloning. Analysis of Ig gene sequences. Nucleotide sequences were aligned to EMBL/GenBank and current databases (V-BASE sequence directory, using MacVector 4.0 sequence analysis software; International Biotechnologies Inc, New Haven, CT). Where there was >2% deviation from a germline VH sequence, the Chang and Casali formula_23_ (http://www.bloodjournal.org/cgi/content/full/94/6/1848#B23)

was used to determine whether the replacement mutations had undergone antigenic selection. We have followed the criteria of Corbett at al in assigning membership of the 2 longer D gene families (D2 and D3), but the requirement for 10 consecutive nucleotides of identity are probably too stringent for the shorter D gene families, and we have followed Fais et al in requiring only 7 consecutive nucleotides with no more than 2 differences. We have eliminated DIR segments and "minor" D segments from analysis.

Terry Hamblin	In the immunoglobulin heavy chain genes there are around 300 base pairs - it varies from gene to gene. Since every cell is the same there is no statistical variation; it is a straight count. It is a very labor intensive assay, but it is just a matter of sequencing genes and a lab with this technique in their repertoire should be able to get the right answer. ZAP-70 by flow is much more variable. The three published papers all used slightly different techniques, and several well known labs failed to get the assay to work. I am dubious of everybody's results outside the three labs that have published.
Terry Hamblin 14 April 2005	There are a number of articles authored by Gerrard Tobin and Richard Rosenquist from Sweden that have looked at the use of VH 3-21 in CLL. They find that such patients have the same prognosis whether they are mutated or unmutated. However almost all of these have 3-4% mutations and few oif any have more than 7% mutations. Also the cases that they refer to mostly use the same light chain and the same D and J segment genes. There is some evidence that those patients who use VH 3-21 who don't use this light chain gene and these D and J segments, don't obey the Swedish rule. This was the finding of workers in France, Spain and Italy. It may be that there is an ethnic difference between Northern and Southern Europeans.
Terry Hamblin 14 April 2005	Terry, if one has been pcr- for over 2 years, does that necessarily mean that the genes are mutated, or can a person be pcr- and have unmutated genes. Does the same apply to a zap 70 situation (negative or positive).. There are no data on how long PCR negativity lasts in mutated and unmutated patients except in respect of the German autograft trial where more than 50% of the mutated patients were still PCR negative at 4 years whereas all the unmutated patients had become PCR positive by 4 years. We do not know whether RFC would be more or less effective than an autograft in this respect. Generally speaking ZAP-70+ equates to VH unmutated and ZAP-70 neg equates to VH mutated but there is some discordance depending on the method used for testing ZAP-70.
Terry Hamblin May 2005	I promised that I would comment on apparent discrepencies in the VH mutation test when done in different labs. How the test is done: For those who want a technical description see the small print at the bottom of this letter, but here is a summary As originally done it involved extracting RNA from the leukemia cells and amplifying it with PCR using appropriate primers to get the reation going. In lymphoma you have to compare the RNA from several cells, since there are minor variations between cells (caused by ongoing mutations) and many contaminating normal B cells. The contaminating B cells can also be a problem in low count CLL, but since there is no ongoing mutation in CLL all the tumor cells should be identical. (This is not precisely true, but for practical purposes it is. Occasionally you can pick out a cell that has an extra mutation, but it does not seem to go on and form a sub-clone the way it does in follicular lymphoma). Once amplified the RNA was sequenced and its sequence compared with the 51 possible VH genes available for perusal on the databases from the internet. There are at least 2 databases in current use and they do differ slightly from each other Since the method was published in 1999 we have refined it and we now use DNA which is much more robust and travels better. The problem with DNA is that there are two #14 chromosomes that carry the VH genes and both may be active. It is therefore possible (though unlikely) that the wrong one may be amplified. If we get two bands and both are possible, the we have to revert to RNA. Sometimes people with CLL actually have two unrelated clones of CLL (after all it is a common disease so why shouldn't you get it twice?). So discrepencies are possible with VH gene assays. There could be two clones. There could be both chromosomes active. A cell with an extra mutation in it could be amplified by chance. A different database might be used for comparison. generally when discrepencies occur it is in cases with mutated VH genes, so in terms of prognosis it makes no difference. Then there could be that bugbear of all laboratories, transcriptional error. Putting the wrong name on the bottle. So far in our lab we have not found any discrepencies when we have resequenced samples, but then all our tests are done by an experienced scientist who helped to develop the test and is well aware of the pitfalls.We do the whole testing in duplicate and have always got the same result in both assays. Terry Hamblin Preparation of cDNA and DNA. Blood samples for testing were taken during the past 5 years. Some were tested immediately, while for others, the lymphocytes were cryopreserved and tested later. Because the VH gene signature is believed not to change during the clinical course of CLL, it was deemed satisfactory to determine this at any stage of the disease whether treated or not. The preferred source material was RNA, as this reduces the possibility of amplifying an aberrantly rearranged VH gene; cDNA was synthesized by reverse transcription using an oligo(dT) primer as previously described. Where RNA was not available, genomic DNA was extracted using the QIAmp blood kit (Qiagen, Hilden, Germany). Amplification of VH genes. One fifth to one third of a sample of cDNA was amplified by polymerase chain reaction (PCR) using a mixture of oligonucleotide 5' primers specific for each leader sequence of the VH1 to VH6 families, together with either mixed 3' primers complementary to the germ line JH regions or 3' primers complementary to the constant region. When there was failure to amplify, an alternative mixture of 5' primers specific for framework 1 region of VH1 to VH6 was substituted. In our hands, the VH1 leader primer also amplifies sequences from the closely related VH7 family. In all cases, PCR was performed in a final volume of 50 µL with 20 pmol of each primer, 50 µmol deoxyribonucleotide triphosphates (dNTPs), and 2.5 U Taq DNA polymerase with reaction buffer (Boehringer, Lewes, E Sussex, UK). Amplification consisted of an initial denaturation step of 3 minutes at 94°C followed by 30 cycles of 94°C, 56°C and 72°C for 45 seconds each, with a final extension step of 10 minutes at 72°C. All PCR reactions were performed in duplicate. For each PCR, a control with no added template was used to check for contamination. Sequencing and cloning of PCR products. 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 (Applied Biosystems, Foster City, CA). However, for the first 35 cases, cloning of gel-purified products into pGEM-TA vector was performed. After transformation of JM109 competent cells, clones found to contain an insert of appropriate size by restriction analysis of plasmid DNA were sequenced. A minimum of 5 bacterial colonies were analyzed. In addition, in 3 later cases, direct sequencing was unsuccessful and the sequence was determined by cloning. Analysis of Ig gene sequences. Nucleotide sequences were aligned to EMBL/GenBank and current databases (V-BASE sequence directory, using MacVector 4.0 sequence analysis software; International Biotechnologies Inc, New Haven, CT). Where there was >2% deviation from a germline VH sequence, the Chang and Casali formula_23_ (http://www.bloodjournal.org/cgi/content/full/94/6/1848#B23) was used to determine whether the replacement mutations had undergone antigenic selection. We have followed the criteria of Corbett at al in assigning membership of the 2 longer D gene families (D2 and D3), but the requirement for 10 consecutive nucleotides of identity are probably too stringent for the shorter D gene families, and we have followed Fais et al in requiring only 7 consecutive nucleotides with no more than 2 differences. We have eliminated DIR segments and "minor" D segments from analysis.