Monday, February 28, 2011

From the Barnabas Trust

Whatever you think of the uprisings in the Middle East, some lawless groups have used them to further their foul ends. Christians and church leaders have come under attack in Egypt in recent days. On Wednesday 23 February, church leader Dawoud Botrous was found stabbed to death inside his home in Assuit. That same day, Egyptian armed forces stormed the 5th century St Bishoy monastery in Wadi el-Natroun, 110km from Cairo. Eight Christians were wounded and rushed to hospital. Armed forces stormed the main entrance gate using tanks and a bulldozer to demolish a fence erected by the monastery for protection against lawlessness during the civil unrest in January. One of the monks said, “The army was shocked to see the monks standing there praying, ‘Lord have mercy’ without running away. This is what really upset them... As the soldiers were demolishing the gate and the fence they were chanting ‘Allahu Akbar’"

Uzbekistan is one of the worst countries in Central Asia for religious freedom, and people of various faiths and denominations are regularly imprisoned for their religious activities. Christian leader David (Dmitri) Shestakov was released on 21 January after four years in prison but news has emerged that he has been placed under “administrative supervision”, which includes a curfew and a ban on visiting certain public places. In addition, he cannot leave his home city without prior police permission, and must report to the regional police at least three times a month. Local Christians fear the police will do all they can to bring another case against him and send him back to prison.

Thousands of women dressed in black brought the city of Jos, Plateau State, Nigeria, to a standstill on 1 February as they marched through the streets to protest against the on-going violence in Plateau State. The march took place after a series of attacks on Christian villages including one incident where five people died after a group of Muslims attacked six villages overnight on 27 January. Christians have also come under attack in Bauchi State where a large explosive device was discovered under a pew at a church in Bauchi Town on 30 January. The street was blocked off by police and the device defused by the bomb squad.

A group of militant Islamists went on the rampage in Indonesia on 8 February demanding the death penalty for a Christian convicted of “blaspheming Islam”. The mob set two churches ablaze and damaged a third. Antonius Richmond Bawengan (58) had been found guilty of distributing books and leaflets that allegedly “spread hatred about Islam” in Temanggung, Central Java, and was given the maximum sentence of five years in prison. Around 1,500 Muslims protested outside the courthouse, calling the sentence too lenient and demanding the death penalty. The crowd chanted “kill, kill” as they pelted the building with rocks.

The violence then spread to surrounding neighbourhoods, where the mob cried “burn, burn” as they targeted the churches. One minister, who saw his church go up in flames, was beaten up by the mob, and at least nine people were rushed to hospital with injuries. The protesters also threw rocks and other missiles at police, attacked a school building, and torched a number of vehicles. The violence came two days after three members of a minority Islamic sect were beaten to death by a Muslim lynch mob, who considered them to be heretics. Both these incidents took place as Indonesia started its “interfaith week”, when the country was meant to celebrate its religious diversity.

Hundreds of local Christians took to the streets in protest after police refused to register the suspected murder of a young Christian man in Gujranwala district, Punjab Province, Pakistan, on 6 February. The 24-year-old, who had been married for just eight months, failed to turn up for work as a driver on 5 February because of illness. When he returned to work the following day, he is said to have been tortured and killed by his Muslim employers. They told his father that he had committed suicide. Following the protests, police did register a murder case against three Muslim men, but it remains to be seen whether or not they will face prosecution for the offence.

A Pakistani church leader said that incidents against Christians are increasing at an alarming rate, especially in Central Punjab, with the perpetrators roaming freely after having killed or robbed. He added, “Most of them have not been reported because of the influence that powerful local landowners have in the areas, and the influence of local MPs. They hold police and judges in the palm of their hands... Christians are not safe.”

Sunday, February 27, 2011

Watching and reading

I watched Heaven Knows Mr Allison the other night. It is a two-hander between Robert Mitchum and Deborah Kerr as a marine and a nun trapped on a Pacific Island during the war against Japan. It has a happy ending with Mr Mitchum only scratched and Miss Kerr with her honor intact. It was a typical piece of period hokum that was enjoyable enough though both the leads played sterotypical characters and nothing very surprising happened. It might have been fairly shocking at the time with a mixed sex cave for sleeping in. Today the most shocking thing about it would be the anti-Japanese racism since the Nips were portrayed as semi-comical ineffectual individuals that one could hardly imagine conquering most of the Pacific.

We also watched the latest instalment of Nanny McPhee, which was fun. How we would all wish for a nanny like that to solve our problems! Emma Thompson, who wrote it, is a Left-wing socialist atheist. She obviously has a strong religious empathy, nevertheless. Christians would recognize the Holy Spirit as McPhee's paradigm.

I have also read the latest Terry Pratchett, I Shall Wear Midnight. This is supposedly for younger readers, though the level of humor, complexity and adult themes is as great as in the other Discworld novels. It concerns the teenage witch, Tiffany Aching. Among the topics it covers are cutting the toenails of an old woman who has not had her shoes off in a year, a miscarriage in a 13-year old brought on by a severe beating from her father, attempted suicide, time travel, young love dissipated, joke shops that sell plastic doggie-poo, the responsibilities of leadership, death and bereavement, hiding one's abilities in order to fit in, weddings and funerals, bullying and standing up to bullies, the hidden truths in folk-myths, and prejudice and how it spreads in a community. Such serious subjects (apart from the joke shop) are handled with fun and humor and a delicious use of English. There are now 38 books in the Discworld series; I feel like reading them all again.

Friday, February 25, 2011

CCL3 - a VH surrogate

It is now very clear that signaling through the B-cell receptor (BCR) is extremely important in determining how active CLL is. My original observation that mutated VH genes predict a more benign version of the disease has turned out to be one of the more important discoveries in the history of CLL, giving rise to the realization that perhaps 30% of cases have BCRs that belong to a limited set of stereotypes and therefore many CLLs derive from B cells involved in the same immune response, and, perhaps more significant for patients, the signaling pathways reveal targets for therapy. Already we have trials of agents that block these pathways, like fostanatinib, which blocks spleen tyrosine kinase (syk) activity and PC-32765, which blocks Bruton tyrosine kinase (Btk) activity.

Disease activity in CLL takes place in the tissues, not the blood, and it has been shown that signaling through the BCR cuases the up-regulation and secretion of the chemokines CCL3 and CCL4. These chemokines were previously called macrophage inflammatory proteins 1 alfa and 1 beta (MIP-1 alfa and beta) and function by attracting macrophages and lymphocytes.

There are tests for signaling through the BCR, but they are cumbersome and only suitable for research laboratories. As is well known, VH mutations are little more tricky than the ordinary lab is used to and various people have proposed surrogates. CD38 turned out to be an independent prognostic marker in its own right and ZAP-70 has proved impossible to standardize. The possibility arises that CCL3, which is secreted in large amounts when BCR signaling takes place might be an even more useful prognostic marker than VH genes. Although most mutated VH genes give rise to a BCR that will not signal and unmutated VH genes give rise to a BCR that does, the correlation is not complete and it may well be that it is the signaling that matters not the structure of the BCR. Since it is secreted, CCL could be measured in the blood by a simple ELISA that even a biochemistry lab could do.

To this end the group at MDACC have developed an ELISA to measure CCL3 in the blood of 351 patients with CLL. They have analyzed high levels of CCL3 as a prognostic marker (end point: time to first treatment) in a multivariate study. Interestingly, the prognostic factors that were statistically significant clinical stage, FISH for chromosomal abnormalities, CD38 and CCL3 levels. Both VH mutations and ZAP-70 were squeezed out by this analysis which suggests that it is the signaling rather than the structure of the BCR that matters.

Of course knowing the VH sequence has other value, since it appears that certain stereotypes have their own individual characteristic types of progression, but this is a simple and cheap test that could be a surrogate for VH testing if it can be reproduced by another study.

Virtue Reborn - an introduction

TN Wright is a heavyweight evangelical theologian. Among some evangelicals he is in bad odour for being associated with something called the New Perspective. He himself strenuously denies being aligned with this movement. Although he sees some value in some of its insights, he finds more to disagree with than assent to. I shall say no more about this, but a search of the web will reveal both the pros and cons of his position.

The book, Virtue Reborn, which I have been reading for the past month begins with the assumption that the death and resurrection of Jesus was the beginning of the new creation. We tend to steer away from eschatology (study of the last things) and often accept that when we die we will go to heaven if we meet certain conditions. The general public seems to think that everyone (except perhaps Adolf Hitler and Saddam Hussain) will go to heaven while others think it depends on having led a good life. Evangelicals think the condition is to have put their trust in Jesus, while other evangelicals insist that it is not enough to give verbal assent to such an idea; it must have made a beneficial change in their lives.

What 'heaven' implies is not clear. The popular picture of sitting on clouds in a frilly nightshirt with a harp has no Biblical basis. The Bible talks about a new heaven and a new earth and Wright insists that this new creation has already started 2000 years ago. This has implications for how we should behave as Christians. Many evangelicals will answer the question of how we should behave with one word - evangelize. However, when St Paul talks about the gifts of the Spirit it is clear that some (not all) are given to be evangelists, just as some are given to be pastors and teachers. What are the rest of us to do?

Wright's answer is that we are to develop a Christian character. He wants us to concentrate on the idea of 'virtue'. Much of what I have written recently is about rejecting the idea that you become a Christian by following a set of rules. The Law never saved anyone. The Law merely demonstrates to us how far short we fall of God's standards. "We don't become Christians by struggling with great moral effort to make ourselves good enough for God, but by the work of the Holy Spirit, bringing us to a faith which looks away from ourselves and trusts, instead, in what God has done for us in Jesus Christ." I quote from the preface of the book.

In some circles this 'developing a Christian character' is known as sanctification and it is clearly seen as a work of the Holy Spirit. But let us be clear. Although we may be viewed as 'spotless' by God the moment we are pardoned or justified or washed clean by His blood or whatever metaphor you prefer for that miraculous event of salvation and in that moment were we to die we would be immediately in his presence in 'paradise', those of us still on earth in this present world still have a lot of changing to do. Many Christians are disappointed that after their conversion experience - which may be spectacular - they are still subject to temptation and still liable to succumb to it. Even Christians of many years standing will find that they still tell lies, cheat at cards, gossip, covet next-door's kitchen, indulge in Internet porn, boast, say cruel things, blaspheme, drive their cars aggressively, lose their temper, and in a million ways fall short of perfection. I know these sins are instantly wiped away - if we confess our sins he is faithful and just and will forgive us our sins and purify us from all unrighteousness - but we will still sin again. Just think of the Apostle Peter refusing to eat with the Gentile Galatians, even after that miraculous vision of unclean beasts and after that wonderful speech to the Council at Jerusalem.

Virtue comes from the Latin 'Vir' - a man - and it is the cultivation of a perfect manly character (as in the perfect man created in God's image and placed in the Garden) that is desired. Tom Wright had an Oxford education in philosophy and it is therefore not surprising that he goes back to Aristotle for the idea of virtue. It is this aspect of the book that some might find intimidating, but I would recommend persevering with it. Eventually he has something important to say.

Thursday, February 24, 2011


I've not been blogging much recently as I have not been feeling too well. Apart from the usual side effects I have had a blocked and runny nose with lots of sneezing and a headache, though no sore throat or other symptoms of a cold.

I have not been thinking very deeply, just watching the blue tits and four squirrels squabbling over the bird food we have put out. Apart from them I have spotted several species of dove, a couple of robins, a pair of chaffinches, blackbirds, crows, a jay, a pair of magpies, the odd sparrow and a herring gull. The weather has been a mixture of dull overcast days of drizzle and warmer sunny days. Early Spring has arrived. We have crocuses and daffodils out and the few snowdrops are finishing. The Forsythia is blooming and the first Camellias are out. Magnolias are in full bud and we are expecting quite a show.

We have just finished a 1000 piece jigsaw puzzle which took us the best part of a month (held up by 5 pieces that fell on the floor). I have also set up the track for the electric train set that I had for Christmas and am making a start on the models. I added some more DVDs to my collection. They are a mixture of old films and new, films I missed and some I wanted to see again. They are in no particular order: Park Row, Pulp, Dressed to Kill, The Last Picture Show, Frighteners, Heaven Knows Mr Allison, Borat, Signs, A Serious Man, Waltz with Bashir, District 9, Vicky Christina Barcelona, A Star is Born, A Letter to Three Wives, Scanners, Where the Sidewalk Ends, The Andromeda Strain, Hotel for Dogs, On the Beach, Smiles of a Summer Night, The Invention of Lying, Carrington, Nashville, Stardust, Born on the Fourth of July, A Man for All Seasons, Carlito's Way, Inkheart , The Heiress, Adam, The Wrestler, Local Hero, The Parallax View, and Public Enemies. The only film I have watched recently is Mr Blanding's Dream House.

I have been reading Virtue Reborn by TN Wright, the present Bishop of Durham. It is quite a difficult read. Since a couple of his previous books have been Surprised by Hope and Simply Christian (not Surprised by Joy and Mere Christianity) I think he may see himself as CS Lewis of the Forties brought up to date for the Noughties. But his books are much longer and don't have that easy application to the common man. Still I will review it in detail at a later blog.

Wednesday, February 23, 2011

Is it always wrong to lie?

Is it ever right to tell a lie?

Utilitarians will say Yes, to avoid a bad consequence; Kantians will say, No, truth telling is a categorical imperative. The hard test of the Kantian position is when your friend asks you to hide him because a murderer is chasing him. When the murderer knocks on your door and asks whether your friend is there, should you tell the truth? Utilitarians would say no, the consequence of truth telling would make you complicit in the murder; Kantians would say telling the the truth is sacrosanct since if you become a liar, you are morally compromised. They would recommend a misleading evasion. "I have no idea where my friend is at the moment," would be true since he might be in a closet or under the bed, but also misleading since he is in your apartment. Bill Clinton was a master of the misleading evasion since as a lawyer he was bound to tell the truth. "I did not have sexual relations with that woman."

Some might say that Jesus was a Kantian. "Is it lawful to pay taxes to Caesar?" "Show me a penny." "Whose figure and inscription is that?" "Render unto Caesar the things that are Caesar's and to God the things that are God's"

I would take neither view since the utilitarian position is impossibly compromised by unnatural consequences - Cock fighting rather than opera should be supported from public funds; but the Kantian position does not reflect the real world. We must be aware of the consequences of our actions and not take a Jesuitical position of willing one consequence yet not being brave enough to let our conscience be pricked.

If we lived in a perfect world there would be no murderer at your door and you could tell the truth with impunity. But we live in a fallen world where not everyone buys into the concept of truth telling. Murderers have forfeited the right to be told the truth, just as the Gestapo had in World War II. Clinton told the truth yet he told a moral lie and his motive was to save his skin. Corrie Ten Boom, who hid Jews in Holland told lies yet her motive was to save the lives of others from a tyrannical regime. Who was in the right? Would a misleading evasion have made her lie more moral? By no means! Because her lie was a] putting herself at great risk (she eventually was sent to a Concentration Camp) and b] motivated to save others, it was of great moral value. To be branded a liar by the Gestapo is a badge of honor.

Monday, February 21, 2011

Wisden's best

In the year 2000, Wisden chose 5 cricketers of the century. They were:

Sir Donald Bradman. No doubt about him. He had a test batting average of 99.94, streets ahead of anyone else. Had he not been bowled for a duck by Eric Hollies in his last test innings he would have averaged over 100 - the equivalent of making a century every time he went out to bat in a test match. Seeing as anything over 40 is regarded as a good average, his achievement is astonishing.

Sir Jack Hobbs. He is the only Englishman selected. His claim to fame is that he made 197 centuries in first class cricket, 98 of them when he was over-40 years of age. It was probably easier to score centuries in that era as Patsy Hendren scored 170 and Wally Hammond 167. Of modern day cricketers, Geoff Boycott scored 151 and Graham Gooch 128. But then Hobbs used to score centuries for fun and used to give his wicket away when he reached the magic three figures so someone else could have a go.

Sir Viv Richards. As captain, he led his all conquering West Indies for 50 test matches. In a different era he dominated world cricket in a way that Bradman did, but his test average was 50.23 and he scored 114 centuries, 24 of them in test matches. It was the way he scored his runs that was so impressive. He once scored 189 in an innings against England in a One-Day International.

Sir Gary Sobers. Perhaps the greatest all-rounder who has ever lived. He scored 8032 runs in 93 test matches at an average of 57.78, but also took 235 test wickets and 109 test catches. He bowled fast or slow and even wrist spin, such was his versatility. In 1957 he scored the then highest score in test cricket at 365 not out and in 1968 was the first to score a six off every ball in an over.

Shane Warne. The only one not to be knighted, and probably won't be given the tabloid headlines about him and Liz Hurley. He has taken 708 test wickets, the second most of all time. Clearly Wisden is still not convinced about Muttiah Muralitheran's action, since he has taken 723. Shane Warne's dismissal of Mike Gatting in 1983 by bowling him round his legs was named "ball of the century".

It must have been a difficult choice. Other players who might have run them close include Sachin Tendulkar who is still playing and has scored the highest number of test centuries and one-day international centuries and has a test average of 55.57, Brian Lara, who has the second highest number of test centuries, as well as the highest score in first class cricket at 501, not out, Sir Len Hutton who was the first professional to captain England and in his day made the highest score In test cricket at 364, Wally Hammond who made 4 scores over 300 in first class cricket and was perhaps the greatest cover point fielder of all time, Peter May a great English batsman and captain who won the Ashes several times in the 1950s and captained Surrey to a decade of County Championships, Dennis Compton the most attractive batsman of his era who scored 123 first class centuries including one over 300 and in 1947 scored the greatest number of runs in a first class season. We should not forget Colin Cowdrey and Tom Graveney, stalwarts of a strong England team of the 1950s and 60s who between then scored 229 first class centuries, or Glen Turner, the greatest New Zealand batsmen and one of the few to score 1000 runs in May.

Then there are the all-rounders: Kieth Millar, Sir Ian Botham, Kapil Dev, Imran Khan and Richard Hadlee; the fast bowlers: Wes Hall and Charlie Griffith, Ray Lindwall, Tyson, Statham and Trueman, Lillee and Thompson, Glen McGrath, Malcolm Marshall, Andy Roberts, Joel Garner, Colin Croft and Michael Holding; and the spinners: Jim Laker, Ray Illingworth, Sonny Rhamadin, Bishan Bedi and Muralitheran himself. What about wicket-keepers? Godfrey Evans was the star of my youth and Les Ames must be mentioned since he scored 102 first class centuries. And this is to barely mention the great Australian team that dominated world cricket in the 1990s.

Did Wisden make the right choices? Send your comments if you disagree.

Thursday, February 17, 2011

Alkylating agents for del 11q

I am sure you will recognise the Dohner et al hierarchical model for FISH results in CLL. This graph has been published hundreds of times. It shows del 17p patients doing worse that del 11q who do worse than trisomy 12, while del 13q patients do better than those with a normal karyotype

At ASH 2010 there was an interesting mini-review in the education session entitled Evidence-Based Mini-Review: The Role of Alkylating Agents in the Initial Treatment of Chronic Lymphocytic Leukemia Patients with the 11q Deletion by Wei Ding and Alessandra Ferrajoli in which they advocated the use of alkylating agents in treating CLL with del 11q.

The first thing that needs to be said is that not all patients with del 11q fall into the poor prognosis pot. Whether it is related to the integrity of the ATM gene on the intact chromosome 11 or whether the poor prognosis cases have genes other than ATM involved is not clear, but certainly some cases do rather better than expected. This means that comparisons need to be made carefully. The two arms of a clinical trial might be well matched for del 11q but poorly matched for bad-risk del 11q and we would not know about it.

I need to remind you of the survival curve discovered by Harmut Dohner

The del 11q patients survived for a much shorter period than those without this chromosomal aberration, but it is important to note that these were young patients aged under 55.

When we examine those over 55 the survival difference disappears. There are also more patients in the over-55 group. However, it must be recognized that they were all patients that needed treatment and that there is a hinterland of patients who do not come to treatment that are underrepresented.

Ding and Ferrajoli reviewed three independent studies, The GCLLSG CLL4 trial, the LRF CLL4 trial and the E2997 trial all comparing fludarabine and cyclophosphamide with fludarabine alone, all of which showed a better response rate and progression-free survival, but no difference in overall survival for the combination. The German trial is only reported in abstract form and unfortunately Ding and Ferrajoli do not give a full reference in their paper. It is Stilgenbauer et al, Blood 2008 volume 112 (ASH abstracts book 18th November, abstract 2089 page 727).

They abstracted the data referring to patients with del 11q from the papers. The German CLL4 trial was in patients under the age of 65 and in these there were 68 patients with del 11q. Ding and Ferrajoli report 100% response rate for FC and only 69% response rate for F in del 11q patients, but I am not sure where they get this information since it is not in the published abstract, nor in the 2006 paper by the same authors of an earlier look at the trial. What the abstract does say is that progression free survival (PFS) was significantly shorter for del 11q patients although they did do better with FC than with F alone. The point is that FC was statistically significantly better than F for PFS for all those with unmutated VH genes, normal karyotype, del 11q, unmutated TP53, CD38>7%, B2MG <5, while for overall survival FC was significantly better than F for those with del11q, trisomy 12 and unmutated TP53. Bear in mind that this report has not yet been subjected to full peer review, despite being more than two years old.

The US Intergroup E2997 trial enrolled 278 patients aged between 33 and 86 (median 61, but still younger than the median age for CLL). However, there were even fewer patients with del 11q, only 40. Although as Ding and Ferrajoli reported there was an indication of a longer PFS with FC (25.2 months) than with F alone (14.9) months, because the numbers were so small, this difference was not statistically significant. Nor did it become statistically significant with longer follow up. The authors conclude, “Thus there is no difference in this study between treatment arms for this group of patients with del (11q).”

The British LRF CLL4 trial was one that I was intimately involved with and my colleague David Oscier has published the impact of prognostic markets on the results. This was the largest of the three trials with 781 patients entered into a three arm comparison of chlorambucil (given at its proper dose of 70mg/sq m/month and continued for up to a year) compared with F and FC. There were 112 patients with del 11q. For PFS the adverse prognostic factors were unmutated VH genes, del 11q, B2MG >4, and treatment with either chlorambucil or fludarabine. For overall survival the adverse prognostic factors were age, B2MG >4, and unmutated VH genes but the differences between the treatment arms was not statistically significant. For patients with del 11q there is a statistically significant improvement in survival for patients treated with FC rather than F (47% v 18.5% at 2 years). What Ding and Ferrajoli fail to say is that FC is similarly superior to chlorambucil, an alkylating agent. Also the addition of cyclophosphamide to fludarabine does not lose the adverse impact of del 11q compared to other karyotypes. Since the thrust of their article is the role of alkylating agents in the initial treatment of CLL patients with the 11q deletion, this is a rather startling omission.

Ding and Ferrajoli have also looked at the addition of rituximab to the combination of purine analog and alkylating agent in some phase II trials. The trial between the Mayo Clinic and Ohio State of PCR was a rather strange one. It recruited 64 evaluable patients between the two sites, but there was some considerable heterogeneity between the sites particularly in respect of performance status, Rai stage and bone marrow diffuse infiltration. This may have accounted for quite different response times between the two sites. There were only 13 patients with del 11q in the trial but nevertheless the trial demonstrated that the treatment free survival (TFS) of del 11q patients was similar to the PFS of the whole group of patients (32 months v 36 months) . What they fail to mention is that PFS is generally shorter than TFS, since treatment is seldom started at the first sign of progression.

This trial acted as historical controls for a more recent small phase 2 trial of 33 patients treated with PR, published in Cancer. Unfortunately, I do not have access to the whole paper but they report a TFS of only 7.9 months compared to 32 months for the PCR patients in the previous trial. Since del 11q is extremely heterogeneous it is not really satisfactory to draw conclusions between such small numbers of patients treated in these trials and no statistical examination is possible. The use of historical controls is also very unsatisfactory.

The final evidence that Ding and Ferrajoli adduce comes from a retrospective analysis of the use of FCR at MDACC. There were 69 patients out of 708 with del 11q23, rather less at 9.7% than in other series, rather confirming the general impression that patients seen at MDACC are often less ill than those seen at other centers. Only 40/69 required treatment. Only 9 patients were Rai stage III or IV and only 8 had bulky lymphadenopathy. However, they generally had adverse prognostic markers with 89% having unmutated VH genes and 74% being ZAP-70 positive. The number with B2MG >4 was only 13 and this suggests to me that MDACC prefers to treat bad risk patients rather earlier in the course of their illness than some other centers. A further 8 patients who had both del 11q and del 17p were excluded from analysis.

It was also from this paper that we gather the information that the Herman CLL4 trial reported a 100% response rate for FC and only 69% response rate for F in del 11q patients, This was a personal communication from Dr Dohner to Dr Keating.

This is rather a good paper in Cancer, not least because it recognizes its limitations. “Our study is limited by the lack of testing for acquisition or presence of mutations in the second allele of the ATM gene, its retrospective nature, the short follow up period, lack of a balanced cohort not receiving chemo-immunotherapy, and lack of a uniformly treated patient group. Although 87.5% (35 of 40) of patients were treated with an FCR-containing regimen, 4 patients were treated with immunotherapy (n=3) or chemotherapy alone (n=1), and 1 patient was treated with RCVP. Caution is needed in the interpretation of the results, as some of our patients were treated on protocols based on their prognostic factors. Another weakness of our study is the lack of CT imaging for response assessment.”

Nevertheless, despite these inadequacies they are able to conclude that “an 11q22 deletion was associated with favorable clinical outcomes, despite the high incidence of other associated adverse prognostic factors, eg, ZAP-70, CD38, and unmutated IGHV gene status. Regimens that contain FCR appear to overcome the poor prognosis traditionally associated with this genetic abnormality.” The response rate for del 11q patients was 100% and at 3 years the actuarial survival was 91% and the relapse-free survival was 77%.

At the same time as this paper was being presented at ASH the results of the German CLL8 trial were becoming known. This study confirms that adding R to FC moves patients with del 11q from the high risk to the standard risk group for both PFS and overall survival. With two studies telling us this I think we can believe it.

Some patients have written to me suggesting that an alkylating agent is an essential ingredient of treatment for patients with del 11q. To my mind that is not what the data are saying. Intensification of purine analog therapy by adding an alkylating agent certainly seems to improve response and prolongs PFS for patients with del 11q, just as it does for other categories of patient with different or no chromosomal abnormalities (except for those with del 17p), but it does not move patients from poor-risk to standard risk in the way that adding rituximab does. What remains unknown is whether cyclophosphamide (with all the attendant risks of secondary MDS) is an essential component of FCR. The current trial comparing FR with FCR should give us an answer.

Monday, February 14, 2011

New drugs for old.

A number of drugs are finding new uses. Aspirin was developed as an analgesic, but it has largely supplanted from that role by paracetamol and ibuprofen. Today it is more likely to be used to prevent stroke or other thrombotic conditions. It is also suggested that it might help prevent pre-eclampsia of pregnancy, dementia and colon cancer. Raloxifen is used for osteoporosis after it failed as an oral contraceptive. Prozac flunked its trials as a hypotensive and Viagra didn't work in angina. The monoclonal antibody bevcizumab was developed to treat metastatic colon cancer, but it has found a new role in treating macular degeneration, one of the causes of blindness in the elderly.

Thalidomide, developed for morning sickness is now standard treatment for multiple myeloma. It also works in leprosy. The anti-gout medication allopurinol was originally developed as a cytotoxic drug. Bimatoprost was originally and anti-glaucoma drug but found more use to make eyelashes grow longer! Bromocriptine, an anti-Parkinson's drug, and Colesevelam, a cholesterol lowering agent, have both found a role in type 2 diabetes. Metaclopramide was originally used as an anti-emetic, then to speed up Barium follow-throughs in radiology and now as an anti-migraine medication. Alemtuzumab was originally used for T-cell leukemias and now is poised to make a difference in multiple sclerosis.

Homoharringtonine has been around for a long time as a treatment for CML. Of course it has been overtaken by imatanib and is seldom see these days. A paper has appeared in the 6th January Blood from MDACC demonstrating that homoharringtonine reduces the expression of Mcl-1 in CLL cells.

Mcl-1 is the major anti-apoptotic protein in CLL. It acts by preventing the pro-apoptotic proteins Bak and Bax from disrupting the mitochondrial membrane and thus initiating apoptosis. Strategies to inhibit the effect of members of the bcl-2 family include the anti-sense oligonuclotide, oblimersen, and the BH3 mimetics like Abt-263. Flavopiridol, roscovitine and SNS-032 all target Mcl-1, so homoharringtonine may well have a place in the treatment of CLL.

Saturday, February 12, 2011

Skin lightening

I am often asked how I manage to cover so many different topics on this blog. Regular readers will know that by far the largest number of postings are about CLL and this is because I make a point of distilling most of the articles I read in Blood, B J Haem, Haemtologioca, Leukemia Research and Leukemia into less turgid prose for the benefit of patients. The next commonest category is religion and here I am aided by my devotional reading and the sermons I listen to.

Most of what else I write is inspired by what I read and this includes 4 daily newspapers, the BMJ, Lancet, NEJM and New Scientist and various books that I am working my way through.

For today's post I want to refer to an article in today's BMJ by Professor Yetunde M Olumide, a retired Professor of Medicine from the University of Lagos in Nigeria. It concerns the practice of skin lightening by the use of cosmetics. I suppose everybody was aware that Michael Jackson was unnaturally pale, but I had no idea how widespread is the use of such agents in Africa. A study among students of a tertiary institution in Enugu, Nigeria found that 71.9% of female students and 53.3% of male students were using these 'cosmetics'. Studies in Senegal and Jordan demonstrate that it is a problem not confined to Nigeria.

Professor Olumide says that the practice is fueled by racial prejudice and the idea that black skin is inferior and that someone with fairer skin is more attractive. In Africa, women with lighter complexions are more likely to be used to advertise alcohol, toiletries, cosmetics and clothing. Highly visible women in the entertainment industry bleach their skin. The message is being sent out that a light complexion equates with beauty and success. Users of these 'cosmetics' believe that they enhance self-esteem and make their prospects for employment and marriage greater.

Skin lightening creams alter the chemical structure of the skin by inhibiting the synthesis of melanin. Active ingredients include hydroquinone, mercury and highly potent fluorinated steroids like betamethasone and fluocinonide. Government regulatory agencies have tried to ban the use of these chemicals, but manufacturers have circumvented the regulations by introducing new chemicals of unknown safety such as niacinamide, oxybenzone and triethanolamine. Some products do not have a list of ingredients no place of manufacture in the label and there are many cheap, substandard or misbranded products easily available. Outside Africa the demand for these products is high. They are often exported by smugglers and are usually found only in local shops where ethnic foodstuffs are sold or in ghettos colonised by black Africans.

Does any of this matter? Yes; because they cause serious ill health. Exogenous ochronosis is particularly associated with the use of hydoquinone. It presents as a dirty, grey-brown, waxy pigmentation on sun-exposed skin. Colloid millium is a transluscent, cream-colored papule 1-5 mm in diameter. Lesions coalesce into nodules or plaques. Other features are of a rotten fish odour caused by the excretion of trimethylamine in the breath, urine, sweat and saliva and the typical steroid effects of skin thinning and infection.

Big pharma is undoubtedly well aware of the use of their products, especially steroid creams, as cosmetics (they should be marketed only as drugs) but they are undoubtedly a nice little earner, so a blind eye is turned. The Nigerian Food, Cosmetics and Drugs regulatory Agency have prohibited the manufacture of cosmetics containing hydroquinone and mercury, but Professor Olumide questions the wisdom of their approving something known as Venus Skin Toning Cream. Generally attempts to reduce the use of these skin lightening have been unsuccessful, and like most areas of public health a change of public attitude has to be engineered to change practice. I suppose some of my readers will think that it is up to the individual to safeguard his own health. But if the public are being lied to, someone should expose the lie.

Friday, February 11, 2011

Bendamustine: is it better?

Bendamustine or Treanda is being seen as the new player on the block by many oncologists and is often regarded as the place to go when the patient becomes resistant to fludarabine combinations.

As can be seen from the structural diagram, Bendamustine is an alkylating agent very similar to chlorambucil with the interpolation of a benzimidazole ring. Enthusiasts have suggested that this gives it properties similar to fludarabine, and have suggested that it will bear some resmblance to fludarabine plus cyclophosphamide when given to patients.

I have yet to see a study which demonstrates that it has purine analog-like properties, indeed Wikipedia (for what it's worth) says Bendamustine acts as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases. However, experts draw attention to this paper to suggest that it has a different action to other alkylating agents. In what follows I have attempted to precis their findings, often using their own words, but often going behind what they report.

The authors assert that the DNA single- and double-strand breaks caused by bendamustine are more extensive and significantly more durable than those caused by cyclophosphamide, cisplatinum, or carmustine. They say that the DNA damage mediated by alkylators has been associated with a regulated form of necrotic cell death whereas Bendamustine as a single agent or in combination with other anticancer agents has also shown proapoptotic activity in several in vitro tumor models.

The study used large-scale screening technologies, including the NCI In vitro Cell Line Screening Project (IVCLSP) and gene microarrays to describe potential mechanisms of action of bendamustine that might distinguish it from other alkylators. The IVCLSP program screens up to 3,000 compounds annually for potential anticancer activity. This program uses 60 human tumor cell lines representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. In a two-stage screening process, compounds are tested for growth-inhibitory activity in all 60 cell lines at a single dose of 10 μmol/L, and compounds that achieve a threshold activity are retested in all 60 cell lines in a five-dose screen. The growth-inhibitory activity of a tested compound is expressed as log (GI50, TGI, or LC50), where GI50 is the concentration required to inhibit tumor cell growth by 50%, TGI is the concentration causing total growth inhibition, and LC50 is the lethal concentration at which 50% of cells are killed. For each compound tested, 60 activity values (one for each cell line) make up the activity pattern, or fingerprint, of the compound. A Pearson correlation coefficient (PCC) of >0.8 indicates >65% agreement in the sensitivity patterns of two compounds and a high likelihood of a common mechanism of action.

The IVCLSP screen was performed for three alkylating agents: melphalan, chlorambucil, and the active metabolite of cyclophosphamide. The sensitivity patterns were shown to be similar for 25 compounds when compared to melphalan (PCC >0.839), 25 compounds compared to chlorambucil (PCC >0.839), and 23 compounds compared to the active metabolite of cyclophosphamide (PCC >0.800). The agents that matched most closely with these drugs were all alkylating agents. Direct comparisons among cyclophosphamide, chlorambucil, and melphalan showed strong correlation coefficients (0.76-0.93).

In contrast, the sensitivity pattern of bendamustine did not strongly correlate with any of the compounds tested. In fact, of the top six matches, only dacarbazine (another alkylator) showed a sensitivity agreement (r2) exceeding 50%. The remaining matches for bendamustine included a topoisomerase 1 inhibitor, an anthracycline, and an anti-metabolite, in addition to two other alkylators, including melphalan. Unfortunately they did not report any results obtained with the purine analogs, and the anti-metabolite was Fazarabine or Ara-AC. These results suggest that bendamustine has a unique mechanistic profile compared with most conventional alkylators, though the correlation with dacarbazine, the alkylator favored for the treatment of melanoma, was 0.792, which was greater than the correlation between chlorambucil and cyclophosphamide (0.762) and cyclophosphamide and melphalan (0.765)

To define the differences in molecular mechanisms of action between bendamustine and other alkylators, they conducted gene expression analyses. Affymetrix GeneChip analysis was used to compare the expression levels of >12,000 genes in SU-DHL-1 cells, a non–Hodgkin's lymphoma cell line, after 8 h of treatment with bendamustine, the active metabolite of cyclophosphamide, chlorambucil, or no drug.

Compared to the control, the genes showing the most significant modulation in the bendamustine experiments were in the following major “functional groups”: (a) response to DNA-damage stress; (b) DNA metabolism; (c) cell proliferation; and (d) cell regulation. A notable difference in the experiments with the active metabolite of cyclophosphamide was the absence of several biological processes belonging to the “DNA metabolism, DNA repair, transcription” and “cell cycle, mitotic checkpoint” groups. Chlorambucil was only highly linked to two biological processes: regulation of biological process and regulation of cellular processes. In a head to head comparison between chlorambucil and bendamustine, two main pathways were identified by this comparison as statistically different between the two compounds: nucleic acid metabolism and mitotic checkpoint-cell cycle regulation. The biological processes with the highest P value in this comparison were deoxyribonucleoside triphosphate metabolism, deoxyribonucleotide metabolism, and regulation of CDK activity.

Many pro-apoptotic genes known to possess p53-response elements in their promoter regions, and considered to be p53-dependent, were found by the microarray analysis to be induced by bendamustine. Examples of these genes are p21 (Cip1/Waf1/cyclin-dependent kinase inhibitor) and NOXA . Both genes were also induced by equitoxic concentrations of the active metabolite of cyclophosphamide and chlorambucil, but to a much lower extent. Bendamustine, but not cyclophosphamide or chlorambucil, caused an appreciable increase in the protein expression of Bax.

In addition to p53-related genes, other regulated genes related to apoptosis and identified in the top 100 modulated genes were four members of the tumor necrosis factor–receptor superfamily. Several of these genes have been shown to play a critical role in the regulation of the extrinsic apoptotic pathway.

Bendamustine induced a stronger (2.5-fold) up-regulation of the DNA repair gene exonuclease-1 (EXO1). EXO1 expression compared with that observed with cyclophosphamide (1.5-fold) or chlorambucil (1.8-fold). DNA-repair capacity has been shown to play a critical role in resistance to alkylating drugs and it may be postulated that these pathways may contribute to the different activity/resistance profiles observed for bendamustine versus cyclophosphamide and chlorambucil.

Bendamustine also caused a significantly greater increase in the proportion of cells in the S-phase of the cell cycle (∼60%) compared with chlorambucil (45%) and cyclophosphamide (37%), based on the DMSO control (37%). It is possible that a defect in mitotic checkpoints inhibits the “physiologic” arrest of the DNA alkylator–treated cells, required for efficient repair of DNA damage before cells are allowed to enter mitosis. Cells entering mitosis with significant DNA damage are reported to result in activation of the death pathway known as mitotic catastrophe. Mitotic catastrophe is a necrotic form of cell death that occurs during metaphase and is morphologically distinct from apoptosis. It can occur in the absence of functional p53 or in cells where conventional caspase-dependent apoptosis is suppressed.

To determine whether bendamustine can cause mitotic catastrophe, it was necessary to find a model in which the apoptotic effects of bendamustine could be distinguished from the potential mitotic catastrophe end point. To this end, bendamustine was tested in cell lines with deficiencies in apoptotic pathways and in the presence of an inhibitor of classic apoptotic pathways. Microscopic analysis of nuclear morphology using 4′,6-diamidino-2-phenylindole staining revealed an increased incidence of chromatin condensation and multinucleation/micronucleation, hallmarks of mitotic catastrophe, in the cell lines tested. Twenty-six percent of the bendamustine-treated cells showed micronucleation compared with only 6% in DMSO control cells. These data indicate that in addition to inducing apoptosis, bendamustine may cause mitotic catastrophe. No results are given for other alkylating agents in this test system.

What are we to make of these results? The first thing that we should note is that the results were produced in the laboratory of Salmedix, a company that has been acquired by Cephalon, who are marketing Treanda in the US. I shall comment later on Big Pharma when I get to discussing the clinical trials that have been done, but I am suspicious of a company that markets a 40-year-old drug at such an inflated price.

Second, were I refereeing this paper, I should have asked for extra data, particularly asking for similarities or not between bendamustine and fludarabine in the IVCLSP assays, and for comparisons with other alkylating agents in the mitotic catastrophe experiments.

Third, these results are all very indirect. Upregulation of genes may or may not exert a pharmacological effect. As far as I know there is no clinical study that shows that bendamustine is effective in TP53 deficient CLL as might be expected from these results. It may be that the combination of mechanism makes bendamustine a more efficient alkylating agent than chlorambucil, but with effectiveness against tumor cells comes toxicity against normal cells. Clinical studies do show more toxicty than chlorambucil.

Fourth, nothing in this study suggests that bendamustine behaves like fludarabine, for all it possesses a benzimidazole ring.

Fifth, I feel I must reiterate my criticisms of the pivotal trial that allowed the FDA to grant approval for bendamustine.

319 patients were randomly assigned to receive either Bendamustine or Chlorambucil (162 Bendamustine, 157 Chlorambucil). The overall response rate was 68% for Bendamustine and 31% for Chlorambucil. CRs were 31% for Bendamustine and 2% for Chlorambucil. Median progression-free survival was 21.6 months for Bendamustine and 8.3 months for Chlorambucil. Bendamustine seems astonishingly better than Chlorambucil, though this does seem a very poor result with Chlorambucil, compared with, say, the LRF CLL4 trial, where the median progression free survival was 22 months.

Toxicity was worse with Bendamustine. Grade 3 or 4 adverse events occurred in 40% for Bendamustine and 19% for Chlorambucil.

This paper is published in JCO which probably means it was turned down by Blood. The criticism which any Blood reviewer would have made (and I have reason to believe was made) would have been that the dose of chlorambucil was too low. They have answered this criticism by declaring that the dose of chlorambucil was equivalent to 60 mg/meter squared which compares well with comparisons with fludarabine (Rai = 40), alemtuzumab (Hillmen = 40) and FC (Catovsky = 70).

That seems to be alright, but then I read the paper more carefully. The two drugs were given according to quite different formulae. Bendamustine was given in a dose of 100mg/sq meter for two days every 4 weeks, while Chlorambucil was given in a dose of 0.8 mg/kg for two days every two weeks. It seems strange that two different calculations were used and even stranger when I see that rather than weighing the patients they used something called Broca's normal weight.

I didn't have a clue what Broca's normal weight was and I am pretty sure that most people reading the paper won't have either. So I Googled it. It turns out to be the height in centimetres minus 100. Does this give the same answer as weighing? By no means. I am 176cm high so my Broca weight is 76kg. Alas my scales make me 90 kg.

If I calculate the dose of chlorambucil I would have got under the LRF4 formula it would have been 148 mg, but under the Bendamustine paper formula it would have been 123 mg. I'm afraid my ideal weight is a little less than my actual weight. For my mother the discrepancy would have been even more. She is only 5ft 3, but weighs about the same as me. The Bendamustine paper would have given her 96 mg of Chlorambucil while the LRF4 calculation would have given her 139 mg. Since most people today weigh considerably more than their ideal weight, it looks to me that the chlorambucil dose given in this trial was too low for a fair comparison.

Since I wrote about this, it has emerged that to get the full benefit from chlorambucil it needs to be given for longer than 6 months- preferably up to twelve.

My conclusion then is that Bendamustine is just a way of getting adequate doses of an alkylating agent into a patient, but I am prepared to be convinced that is more than that if soeone would only show me the data.

Thursday, February 10, 2011


Regular readers will have notice that I usually use American spelling rather than British spelling on this blog. The reason is that the majority of my 300-500 daily readers are American. I think that the different spellings are pretty well understood whichever side of the Atlantic one comes from, but I have been asked about 'ise' and 'ize' endings. In America everybody uses 'ize' but either is acceptable in Britain. The Times, the Oxford University Press and the Cambridge University Press all prefer 'ize' but all the other newspapers and most other publishers use 'ise', perhaps taking their lead from Shakespeare. In King Lear, Kent exclaims, "Thou whoresome zed! Thou unnecessary letter!"

The ultimate source of the ending is the Greek '-izein'. If the word comes to us from Latin it is from '-izare'. For the 'ise' ending we must blame the French who adopted 'iser' for verbs from these origins.

There are, however, a whole chunk of words whose origins can never be traced to the Greek '-izein' which must always be spelled 'ise'. Here is as complete a list as I can muster: advertise, advise, apprise, chastise, circumcise, comprise, compromise, demise, despise, devise, disfranchise, enfranchise, enterprise, excise, exercise, franchise, improvise, incise, premise, revise, supervise, surmise, surprise, televise.

It is because the list is hard to remember that some people adopt the universal 'ise'. It is not that hard to remember. The 'vise' words generally come from 'videre' to see; the 'cise' words come from 'cidere' to cut, the 'mise' words from 'mittere' to send and the 'prise' words from 'prehendere' to take hold. 'Despise' comes from 'specere' to look (down on). The 'franchise' words come from the French (naturally) and therefore are 'ise'. Advertise and chastise are the remaining two and those you have to remember.

Wednesday, February 09, 2011

Law and Grace; Galatians 3:26-4:11

Immigration is today's hot potato. Jeremy Clarkson, the presenter of Top Gear who has made a profession of being non-PC, recently made a crack that the Mexican Olympic team never wins medals because anyone who can run, jump or swim is already in the USA. The British Prime Minister, David Cameron, has declared that multiculturism is dead. Mihr Bose, recently the BBC sports editor and himself of Indian origin, has said that he can't understand why Bengalis should want to come to England if they don't adopt British culture. If they want to continue with Bengali culture, why not stay in Calcutta where it isn't so cold and wet?

On the other hand we are very aware that the color of one's skin used to determine how one was treated. I remember Bed and Breakfast hostelries with signs in their windows that said, "No pets, no coloureds, no Irish". Even though we have a female monarch, and have had a female Prime Minister, as well as female Presidents of the Royal College of Pathologists and the Royal College of Physicians, there is still a glass ceiling for women in business and we have never had a female leader of the Labour Party, that well known egalitarian organisation. Slavery was abolished in Britain and the US many years ago, but it persists in the Arab world and in many parts of Africa, while Jim Crowism continued in America until the time of Lyndon Johnson.

If anything, things were worse in Biblical times. The Jews prided themselves as being 'children of Abraham' and called the heathen nations 'swarms' or 'hordes' or even 'dogs'. It was not one-sided. The Romans looked down on the Jews. Aristotle called slaves 'animated implements' or a 'tool that breathes', while Josephus states that 'the woman, so says the law, is inferior in all things to man'.

So it is a radical idea that Paul espouses when he says in Galatians 3:28, "There is neither Jew nor Greek, slave nor free, male nor female, for you are all one in Christ Jesus." Galatians 3:26.

Where does this unity come from? From Christ. "You are all sons of God through faith in Christ Jesus, for all of you were baptized (or immersed) into Christ."

This doesn't mean that we are all the same. In China, the cultural revolution under Mao had brain surgeons cleaning the operating theater floor and cleaners doing brain surgery. Yesterday, Lord Lang, a member of the House of Lords, declared that "Bus drivers, waitresses and other people in ‘unimportant’ jobs are not fit to sit in judgement on the business interests of former ministers". Now this sounds like arrogance and snobbery of the first order, and I am sure that this fellow is a thoroughly nasty man, but there is a nub of truth in what he said. Certain tasks require training and education and not everyone is capable of succeeding in every task. I would hate to see my performance on Dancing on Ice.

It is not our nature or genetic endowment, or even our behavior that makes us all one; it is the fact that we have been immersed in Christ. As Paul puts it we have 'clothed ourselves with Christ'. It is as though when God the Father looks at us we have as it were Harry Potter's invisibility cloak around us and on top of that the cloak of Christ, so that he looks at us and sees his Son.

Paul concludes this chapter by telling us that we are Abraham's seed and heir according to the promise. This is the consequence of being clothed in Christ. It is not just that we avoid Hell, escape the consequences of our sin. It is not just a 'Get out of jail free' card. We have become co-heirs with Christ. Did you know that we will judge angels? We will share in the glory that is his. Ephesians 1:13,14 tells us that we have already had the first installment of that glory now, with the gift of the Holy Spirit. He is the deposit with us of glory to come; the guarantor of glory.

I have preached on Galatians chapter 4 at Christmas time. "When the time had fully come, God sent his son, born of a woman." All very true, no doubt but wrenching it out of context. The first 7 verses of chapter 4 are an illustration of what has gone before. He takes up the story of a pedagogue, a slave in charge of the children of the family, who has absolute authority over them, even though one day, the heir would have authority over him -an animate implement - as Aristotle called him. The tutor-slave could whip the heir if he didn't learn his lessons. I suppose there have been future prime ministers going through the English Public Schools system who suffered bullying by prefects and house masters (read Tom Brown's Schooldays) but who later enacted laws to inflict even the death penalty on citizens.

Paul is making the point that when Israel was immature it had to be treated like a child, being subject to rules. I remember being caned at school. There were school rules and by the age of eleven I thought some of them were silly. A few of us deliberately disobeyed one of the rules, flagrantly doing so in front of the headmaster. For this we were punished. We were under the Law and had to take the consequences. When I graduated to senior school, that particular rule no longer applied. I was considered mature enough to make my own decisions.

In my Christmas sermon I talked about the 'fullness of time' referring to the pax Romana meaning that the spread of the Gospel was facilitated along straight Roman roads in the Lingua Franca of the time - Greek. But this is not what is meant at all. What it signifies is a time of maturity. The recourse to the Law's governance is over. What was it that made the difference? It was the coming of Christ.

God sent of his Son - he was divine. Born of a woman - he was human. Born under Law - how else could be fulfil the Law's demands? To redeem those under the Law - how else could he be our surrogate? That we might receive the full rights as sons. Now the telios - the completeness is achieved.

This does not mean we are now perfect. In the famous chapter on love, 1 Corinthians 13, Paul tells is that when perfection comes the imperfect disappears. The imperfect is with us still, I am afraid. The Warfield position is that miraculous gifts have ceased because we have Scripture and they date this 'perfection' from the confirmation of the whole canon of Scripture. I once held that position but I do so longer. Not that I have been a witness to miracles, nor do I speak in tongues or foresee the future, but it seems to me clear from Scripture that the perfection referred to is the new creation, the new heaven and earth when Christ returns. In the meantime the Kingdom has begun, we are regarded as adults, we have the deposit of the Holy Spirit, the New Covenant of Jeremiah 31:33 is in operation, we are no longer slaves but sons and heirs (v 7).

The Galatians had been slaves to pagan gods who by their nature were no gods. 'On Mount Olympus's towering tops, some Greeks and Romans picked some hops' was a mnemonic we learned in order to remember the order of the cranial nerves. Not much call for Mount Olympus, these days, but plenty of pagan gods to worship. Most commonly it is money, but sports stars, actors, pop-singers and 'celebrities' are all prominent. Fame, success, family, houses, cars, holidays; all by their nature no gods, yet worshiped as if they were. Can you identify a god that enslaves you?

But if you are a Christian you know God - or rather you are known by God. I think the most terrifying passage in the whole Bible is from Matthew 7.
"Not every one that saith unto me, Lord, Lord, shall enter into the kingdom of heaven; but he that doeth the will of my Father which is in heaven. Many will say to me in that day, Lord, Lord, have we not prophesied in thy name? and in thy name have cast out devils? and in thy name done many wonderful works? And then will I profess unto them, I never knew you: depart from me, ye that work iniquity."

But Paul tells us that as Christians we are known by God.

It is said that when the slaves in Jamaica were freed, the next morning they rose early and made their way to the sugar cane fields, for they knew no other way. Paul castigates the Galatians, "How is it that you are turning back to those weak and miserable principles?" Martin Luther in illustrating this verse, turned to Aesop's Fables. The dog with a joint of meat in his jaws sees his reflection in the lake. He opens his mouth to grasp the reflected meat and in so doing drops the joint in the water. Aesop's point was the stupidity of greed; Luther's point was the stupidity of stupidity; why reach for a reflection when you have the real thing? By taking up the Law, the Galatians were snatching at shadows. Do we do the same?

Paul tells us in verse 11 that he feared for the Galatians. Let me ask you this question. You do not commit adultery. Is it because you fear the consequences of the Law or because you love the Lord? You do not steal. Many do and they are seldom caught. They say that only 3% of crimes are detected. You could definitely enhance your living standard with a little well tempered theft with very little chance of being caught. But you are a Christian and Christians don't steal. Why not? Is it because God's all seeing eye will spot your transgression and there will (literally) be Hell to pay? Or is it because you love the Lord and wish to please him.

When you were a child you used to obey your parents. Why was that? because if you didn't they would thrash you? Or shout at you? Or was it because you loved them and wished to please them?

Tuesday, February 08, 2011

Cats of various sorts

Cats: the world, the flesh and the Devil.

We called her Kitten – just a farm cat loaned
for us to nurse with chloramphenicol
and food; for cats are never really owned
the way that dogs are or a Barbie doll.
She used to bring us gifts: a mouse’s head
or tail, but then she left. No debt was owed.
Her present world attracted her instead,
or else another pet killed on the road.

Beware the tigress, prowling and feeding deep
in the forest, needing her yellow stripes to steal
from her black back the drops of sun that seep
through darkness. Death is her dominion; feel
the blood congeal upon those amber teeth.
The taste of every future meal she knows.
She longs for flesh. Light fails to pierce beneath
the trees, so nothing shows and chaos grows.

The lazy lion roars a lofty yawn;
he lifts his paw and shakes his tawny mane
and contemplates his pride with studied scorn.
In certain ease the lord of air and plain
enjoys his comfort and his prey’s distress;
views his domain; displays his solemn power.
Vain, indolent and proud, he nonetheless
considers whom he might today devour.

Monday, February 07, 2011

Under the governess: Galatians 3:19-25

Jane Eyre was that stalwart of Victorian fiction - a governess. She had control over the children of the household. No doubt the oldest son would one day inherit the whole pile, but while he was under her tutelage her word was law. He might be master one day, but for today she was mistress. Her position was like that of the pedagogue of Galatians 4:2; a servant yet still in control of the heir or in Roman times - a slave. Paul is telling us that the law is like a governess Galatians 3:24 put in charge of us until we reach maturity.

In verse 19 Paul asks rhetorically, "What then is the purpose of the Law?" The first answer he gives is because of sin. in Romans 4:15 he writes that where there is no law there is no transgression. It is the Law that holds up a magnifying glass to sin. Look at a beige carpet. It looks fine; no blemishes. But examine it with a magnifying glass, Sherlock, and you will see the stain where last week you spilled the tea, the slight dab of tomato ketchup when you ate your supper on your knee because you were too lazy to lay the table; the speck of mud when you didn't wipe your boots properly and the posit of vomit from when your baby granddaughter visited.

I didn't know what coveting meant until I read the ten commandments. I certainly didn't know it was wrong. If that were not enough, the Sermon on the Mount is like an electron microscope on sin. It magnifies ten thousand times! It's not just murder or adultery but the attitude of the heart that counts.

The second reason that Paul gives is that the Law is temporary. It was added until the Seed that was promised had come. In other words it lasted until Jesus, the Messiah, came.

My wife had her wisdom teeth out when she was 20. Still her father had to sign the consent form. In those days you had to be 21 to be an adult. Today in the UK we have something called Gillick competence. It arose because Mrs Gillick thought that 14 was too young for her daughter to be on the pill. Learned judges decided that young girls could decide that at an early age. Seems to me that it gives a license for pedophiles, but the age of maturity has been known to be different for different things; 14, 16, 18, 21? Paul marks the age of maturity with an event; the coming of the Messiah. Things are different now; the Law is written in our hearts.

There is a mistranslation in verse 24. Both the KJV and older editions of the NIV have it wrong. The Law was not put in charge to lead us to Christ. There is no way that the Greek can be so translated. The marginal note in the older NIV has been incorporated in the text of newer edition. The law was put in charge until Christ came.

There is also a difference between the old agreement and the new. The Old Covenant was conditional; God would do this, if Israel did that. It is said that there were two tablets of stone the way that we have two copies of a contract; one for each party. The New Covenant is unconditional. It depends on one party only (verse 20). God promises this. There is no promise on our behalf. If we sin we are not condemned. If we sin we are forgiven. Amazing but true. How can it be? Because our sin was already punished on the tree. We may be rubbing salt in the wounds of the Savior, but nevertheless we are forgiven.

Paul asks a second question, "Is the law opposed to the promises of God?" The answer is a definite 'No'. Absolutely not!. If the law could have imparted life it would certainly have done so. It was pointing in the right direction. But it had no power to do so (verse 21). It fizzled out. Instead what was promised, given through faith in Jesus Christ, is life, and eternal life at that (verse 22). The law was like a prison. We were held fast by its precepts. Imagine a man sitting on a chair, his arms on the armrests. Here is a simple phrase of the law and ... SNAP! a manacle clamps down on his wrist. Another word from Leviticus and his other wrist is made fast. Here is an undiscovered clause from Deuteronomy and his feet are fettered. If thou should mark our iniquities. who could stand? The whole head is sick and the whole heart is faint. From the sole of the foot even to the head, there is no soundness in it, but bruises and sores and raw wounds. The heart is deceitful above all things and exceedingly corrupt; who can know it. The Law is all diagnosis and no treatment.

We were in prison then ... My chains fell off! I was free!

Now that faith has come, we are no longer under the supervision of the law (verse 25).

array CGH

When I started as a hematologist, I worked for a guy, who though a hematologist by name, was not much interested in patients with leukemia, sickle cell disease, hemophilia or hemolytic disease. His main interest was in chromosomes. He would take a weekly trip to a hospital for the mentally handicapped to take blood from what he then called FLKs (funny looking kids) in order to recognize strange chromosomal disorders. He is long dead now and I can't find any publications under his name on PubMed, but he got me interested in chromosomes, and I remember a case of CLL who developed MDS with trisomy 8 that I diagnosed while working for him. Of course I am only assuming that it was trisomy 8 because in those days chromosomes could only be distinguished by their size and shape and chromosomes 7-12 all looked the same - they were called 'C'-group chromosomes.

It was not until chromosome banding came into being that we could number the chromosomes accurately. The first type of banding was introduced by Janet Rowley in Chicago, but this required a fluorescent microscope to recognize the quinacrine staining. The much more useful trypsin/Geimsa banding was discovered by my former colleague Marina Seabright shortly afterwards following a laboratory accident when trypsin was spilled onto a chromosome spread. This is now the standard method, but it does suffer from difficulties in spotting very small deletions and additions. This upside down slide shows del 5q, 13q and 20q by trypsin banding.

Readers will be familiar with FISH or fluorescent in situ hybridization, a technique which allows the detection of missing of additional genes, but this will only be of value if you know which gene you are looking for. The picture shows another use, the detection of translocations. Here we see the t(11;14) translocation typical of mantle cell lymphoma. In some of the cells the red and green probes are fused and appear yellow.

There is also spectral karyotyping which is a molecular technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors.
Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes. Any translocation, even though quite small can be visualized.

Comparative genomic hybridization (CGH) is the latest method for measuring small cytogenetic aberrations. In a typical CGH measurement, total genomic DNA is isolated from test and reference cell populations, differentially labeled and hybridized to metaphase chromosomes or, more recently, DNA microarrays. The relative hybridization intensity of the test and reference signals at a given location is then (ideally) proportional to the relative copy number of those sequences in the test and reference genomes. If the reference genome is normal, then increases and decreases in the intensity ratio directly indicate DNA copy-number variation in the genome of the test cells.

Data are typically normalized so that the modal ratio for the genome is set to some standard value, typically 1.0 on a linear scale or 0.0 on a logarithmic scale. Additional measurements such as FISH or flow cytometry can be used to determine the copy number associated with a given ratio level. In the picture there is reduplication of the short arm of chromosome 5 plus other fine aberrations which could only have been detected by high resolution CGH such as this.

The latest use of CGH in CLL comes from Harvard where they have compared familial and sporadic CLL. Familial cases had fewer cases with loss at 11q and more cases with a gain of material near the centromere at 14q.

siRNA knockdowns of ROR1 and FMOD

Small interfering RNAs (siRNAs) are a useful tool in finding out what genes do. Previously it was necessary to produce genetically engineered mice with particular genes 'knocked out' (knockout mice) in order to find out what genes did.

siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science.[Hamilton A, Baulcombe D (1999). "A species of small antisense RNA in posttranscriptional gene silencing in plants". Science 286 (5441): 950–2.] Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl, and colleagues in a paper published in Nature.[Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494–988.] This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development. To me they sound like Nobel Prize candidates.

siRNAs have a well-defined structure: a short (usually 21-nucleotide) double strand RNA (dsRNA) with 2-nucleotide 3' overhangs on either end: Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.

This technique has been used to look at two genes in CLL by Mellstedt's group in Sweden. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B-cells. It is located on chromosome 1p31.3 and codes for a 105 kDa protein. It is normally expressed in heart, lung and kidneys. It binds to Wnt5a in CLL cells.

Fibromodulin (FMOD) is a 59 kDA collegen binding protein coded for by a gene at 1q31.2 and is found in many types of connective tissue including cartilage, tendon, skin, sclera adn cornea. It is known to bind to TGF-beta.

Using siRNA knockdown the FMOD and ROR1 proteins were significantly reduced in CLL cells, and increased apoptosis occurred in CLL cells but not normal B cells.

Two more genes involved in apoptosis and possible targets in CLL.

Acadesine - a new agent in CLL

I have been involved peripherally with Acadesine for several years now. It had the advantages of being a purine analog that didn't kill T cells and that had already been trialed (albeit at a lower dose) in over 1000 cardiac patients. A paper in Blood last October demonstrates that its activity in CLL is not, as had been previously thought, confined to its activity against adenosine monophosphate activated protein kinase (AMPK). There is another mechanism that is independent of TP53 or ATM status. This suggests that Acadesine might be even more useful if it reaches clinical practice.

Sunday, February 06, 2011

LEF1: the Wnt pathway in CLL

A paper from Neil Kay's group at the Mayo looks at LEF-1 which I mentioned in the last article as being part of the canonical Wnt signaling pathway.

LEF1 stands for lymphoid enhancer binding factor 1. They had previously identified aberrant expression of LEF1 in CLL. In this paper they have analyzed the canonical Wnt pathway by gene expression profiling. Whereas the pathway is inactive in normal peripheral B cells it is constitutively activated in both CLL and monoclonal B cell lymphocytosis. This suggests a role for the Wnt pathway in early CLL leukemogenesis. They also found that knockdown of LEF1 by siRNA decreases the survival of CLL cells.

TCF4, which codes for a downstream target of the Wnt pathway is overexpressed in CLL as is another Wnt target gene, CCND2. IGFBP4 protein, a Wnt inhibitor, is also overexpressed in CLL.

An important observation of this study was that LEF1 was not upregulated in activated B cells, though it was in CLL cells of both the mutated and unmutated subsets - demonstrating that it is not simply an activation marker.

Although the precise manner that the Wnt pathway is affected in CLL is not yet clear, it does provide fopr a potential target for CLL treatment. In one case though LEF1 knockdown had no effect on survival; this case had 17p deletion, suggesting that Wnt becomes irrelevant in these cases.

Notch and VEGF pathways are abnormal in CLL and these are known to interact with Wnt signaling.

Finally there is a speculation that the LEF/TCF repressor, VentX, might be lacking in CLL cells.

Saturday, February 05, 2011

Wnt pathway and cancer stem cells.

One of the problems in being retired is that medicine moves on. New techniques, new disciplines and new pathways keep appearing until scientific papers become increasingly opaque. The Wnt genes have been around a little while, but because they have hardly impacted on CLL I have ignored them. However, the last CLL paper I looked at mentioned them so I thought that I ought to try and understand them.

The name Wnt was coined as a combination of Wg (wingless) and Int and is usually pronounced as 'wint'. The wingless gene had originally been identified as a recessive mutation affecting wing and haltere development in the fruit fly, Drosophila melanogaster (halteres are small knobbed structures modified from the hind wings in some two-winged insects; they are flapped rapidly and function as accelerometers to help the insect maintain stability in flight). They can be seen on this picture of a crane fly.
It was subsequently characterized as segment polarity gene (genes whose function is to define the anterior and posterior polarities within each embryonic parasegment) in Drosophila melanogaster that functions during embryogenesis and also during adult limb formation during metamorphosis. The INT genes were originally identified as vertebrate genes near several integration sites of mouse mammary tumor virus (MMTV). The Int-1 gene and the wingless gene were found to be homologous, as shown by similar amino acid sequences of their encoded proteins.

Mutations of the wingless gene in the fruit fly were found in wingless flies, while tumors caused by MMTV were found to have copies of the virus integrated into the genome forcing overproduction of one of several Wnt genes. The effort to understand how similar genes produce such different effects has revealed that Wnts are a major class of secreted morphogenic ligands of profound importance in establishing the pattern of development in the bodies of all multicellular organisms studied.

Wnt proteins are therefore known as secreted morphogens that are required for basic developmental processes, such as cell-fate specification(ie what happens to individual cells in development), progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. There are at least three different Wnt pathways (see figure which will enlarge when clicked):
the canonical pathway, the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway. In the canonical Wnt pathway, the major effect of Wnt ligand binding to its receptor is the stabilization of cytoplasmic beta-catenin through inhibition of the bea-catenin degradation complex. Beta-catenin is then free to enter the nucleus and activate Wnt-regulated genes through its interaction with TCF (T-cell factor) family transcription factors and concomitant recruitment of coactivators. Planar cell polarity (PCP) signaling leads to the activation of the small GTPases RHOA (RAS homologue gene-family member A) and RAC1, which activate the stress kinase JNK (Jun N-terminal kinase) and ROCK (RHO-associated coiled-coil-containing protein kinase 1) and leads to remodelling of the cytoskeleton and changes in cell adhesion and motility. WNT-Ca2+ signalling is mediated through G proteins and phospholipases and leads to transient increases in cytoplasmic free calcium that subsequently activate the kinase PKC (protein kinase C) and CAMKII (calcium calmodulin mediated kinase II) and the phosphatase calcineurin.

In the canonical pathway Wnt proteins bind to cell-surface receptors of the Frizzled (FRZ) family, causing the receptors to activate Dishevelled family proteins (Frizzled and Dishevelled are obviously descriptions of fruit flies when these genes are missing) and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. Cell surface FRZ proteins usually interact with a transmembrane protein called LRP. LRP binds FRZ, Wnt and axin and may stabilize a Wnt/FRZ/LRP/Dishevelled/axin complex at the cell surface (known as the receptor complex). Phosphorylation of the cytoplasmic domain of LRP by CK1 and GSK3 can regulate axin binding to LRP (interaction 1 in Figure). The protein kinase activity of GSK3 appears to be important for both the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axin complex and the function of the Axin/APC/GSK3/β-catenin complex. β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression (interaction 2, Figure).

Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC (Figure) (APC stands for adenomatous polyposis coli, one of the genes that is involved in bowel cancer). The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. Several protein kinases and protein phosphatases have been associated with the ability of the cell surface Wnt-activated Wnt receptor complex to bind axin and disassemble the axin/GSK3 complex. The protein kinase activity of GSK3 appears to be important for both the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axin complex and the function of the Axin/APC/GSK3/β-catenin complex. Phosphorylation of β-catenin by GSK3 leads to the destruction of β-catenin (Figure).

The Wnt pathway is obviously important for morphogenesis and my daughter, who was particularly good at developmental biology, remembers it from her time at Oxford. However, this will not interest most of my readers who will be more concerned with the fact that alterations of Wnts, APC, axin, and TCFs are all associated with carcinogenesis. There are at least 17 members of the Wnt family.

The WNT/ β-catenin pathway is activated by WNT1, WNT3, WNT3a, WNT7a, and WNT8. In the absence of Wnt signaling, β-catenin is destined for destruction. Several genes have now been identified as the target of β-catenin /TCF transcriptional regulation. These include MMP7 (Matrix Metalloproteinase-7), UPAR (Urokinase-type Plasminogen Activator Receptor), CD44, c-Myc, c-Jun, FRA1 (Fos-Related Antigen-1), CcnD1 (Cyclin-D1), PPAR-Delta (Peroxisome Proliferative Activated Receptor-Delta), TCF1 (Transcription Factor-1), Fibronectin , Slug , Gastrin, Cox2 (Cyclooxygenase-2) and the Gamma2 chain of Laminin5. Many of these genes, including CcnD1 and c-Myc have crucial roles in cell growth, proliferation and differentiation, and are inappropriately activated in colon cancer, Burkitt's lymphoma and mantle cell lymphoma as well as many other tumors. c-Myc gene induced by β-catenin may induce expression of p53, and p53 upregulated p21WAF1 and p130/RB2, resulting in growth arrest. Another target of β-catenin is Vimentin, a protein involved in cell migration. Vimentin is a direct target of the β-catenin /TCF transactivation pathway in human breast cancer cells. WNT signaling can prevent apoptosis by up-regulating anti-apoptotic proteins, such as the caspase inhibitor, survivin, and stimulate angiogenesis via up-regulation of VEGF (Vascular Endothelial Growth Factor). Several proteases capable of degrading extracellular matrix, such as Matrilysin/MMP7 and MMP26, as well as cell adhesion molecules such as CD44 and NRCAM (Neuronal Cell Adhesion Molecule) are WNT targets that could aid the tumour cells in invasion and metastasis. The Canonical WNT signaling cascade also regulates the NRSF/REST and ENC1 (Ectodermal-Neural Cortex (with BTB-like domain)-1) genes, thereby controlling the progenitor cells. Cldn1 (Claudin-1) is also involved in the Ctnn-Beta -TCF/LEF signaling pathway, and increased expression of Cldn1 may have some role in colorectal tumorigenesis.

One of the interesting recent developments in cancer has been the recognition of cancer stem cells (CSCs). Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. Last September an investigation was published of reporting an examination of the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres using the human colon cancer cell line HCT-116. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to ≤ 1% in the corresponding parental cells. Additionally, colonospheres showed reduced membrane bound β-catenin but had increased levels of total β-catenin, cyclin-D1 and c-myc and down regulation of axin-1 and phosphorylated β-catenin. Increased expression of β-catenin was associated with a marked transcriptional activation of TCF/LEF. The latter was greatly decreased following down regulation of β-catenin by the corresponding siRNA.

Cancer stem cells that are resistant to conventional chemotherapy seem to be one possible reason that cancer returns after complete remissions. Speculation suggests that this is true not only for epithelial tissues like the colon, but also for leukemias and lymphomas. It may be that therapy targeted to the Wnt/β-catenin pathway will be effective in eliminating this population.