Stories [9]: A Healthy Volunteer

20 09 2010

The host of Next Grand Rounds (Pallimed) asked to submit a recent blog post from another blogger in addition to your own post.
I choose “Orthostatics – one more time” from DB Medical rants and a post commenting on that from Musings of a Dinosaur.

Bob Center’s (@medrants) posts was about the value of orthostatic vital sign measurements (I won’t go into any details here), and about who should be doing them, nurses or doctors. In his post, Bob Center also mentioned briefly that students were seeing this as scut work similar as drawing your own bloods and carrying them to the lab.

That reminded me of something that happened when I was working in the lab as a PhD, 20 years ago.

I was working on a chromosomal translocation between chromosome 14 and 18. (see Fig)

The t(14;18) is THE hallmark of follicular lymphoma (lymphoma is a B cell cancer of the lymph nodes).

This chromosomal translocation is caused by a faulty coupling of an immunoglobulin chain to the BCL-2 proto-oncogene during the normal rearrangement process of the immunoglobulins in the pre-B-cells.

This t(14;18) translocation can be detected by genetic techniques, such as PCR.

Using PCR, we found that the t(14:18) translocation was not only present in follicular lymphoma, but also in benign hyperplasia of tonsils and lymph nodes in otherwise healthy persons. Just one out of  1 : 100,000 cells were positive. When I finally succeeded in sequencing the PCR-amplified breakpoints, we could show that each breakpoint was unique and not due to contamination of our positive control (read my posts on XMRV to see why this is important).

So we had a paper. Together with experiments in transgenic mice, our results hinted that t(14;18) translocations is necessary but not sufficient for follicular lymphoma. Enhanced expression of BCL-2 might give make the cells with the translocation “immortal”.

All fine, but hyperplastic tonsils might still form an exception, since they are not completely normal. We reasoned that if the t(14;18) was an accidental mistake in pre B cells it might sometimes be found in normal B cells in the blood too.

But then we needed normal blood from healthy individuals.

At the blood bank we could only get pooled blood at that time. But that wasn’t suitable, because if a translocation was present in one individual it would be diluted with the blood of the others.

So, as was quite common then, we asked our colleagues to donate some blood.

The entire procedure was cumbersome: a technician first had to enrich for T and  B cells, we had to separate the cells by FACS and I would then PCR and sequence them.

The PCR and sequencing techniques had to be adopted, because the frequency of positive cells was lower than in the tonsils and approached the detection limit. ….. That is in most people. But not in all. One of our colleagues had relatively prominent bands, and several breakpoints.

It was explained to him that this meant nothing really. Because we did find similar translocations in every healthy person.

But still, I wouldn’t feel 100% sure, if so many of my blood cells (one out of 1000 or 10.000) contained t(14:18) translocations.

He was one of the first volunteers we tested, but from then on it was decided to test only anonymous persons.

Related Articles


Evolution and Medicine. Cancer and adaptive immune responses as evolutions ‘within’.

29 12 2008

I had almost finished my submission for the Grand Round when I took a look at the site of the host, Moneduloides*, to find that this edition had “the interface of evolution and medicine” as a theme.

What should I write about, considering I only had a few hours to write about this difficult theme?

Quite coincidentally (or not, considering the forthcoming bicentenary of Darwin’s birth (1809) and the 150th anniversary of the publication of ‘On the Origin of Species’) the December 2008 Lancet is a Special Issue: Darwin’s Gifts. But it would be to easy to just summarize one or two articles from this Lancet issue…

Evolution and Medicine can be interpreted differently. One can just see it as the evolution of medicine. Enough to write about this theme….

One can also see the theme in the light of consequences of evolution on medicine or illnesses. Indeed there are ample examples of the consequences of men’s evolution on the susceptibility to certain illnesses, e.g. see moneduloides’ blog about the consequence of human bipedalism.

Yesterday @carlosrizo (twitter) pointed out a link to Darwinian Medicine 2.0″. Since this would be of special interest to this web 2.0 audience, I took a look to see if I could ‘use’ this blogpost. However the post appeared to be based on a rather distorted interpretation of natural selection. Darwinian Medicine 1.0. is considered synonymous with eugenics (!), whereas Darwinian Medicine 2.0 is “gentler, interested in finding “evolutionary causes and remedies for diseases.” while leaving out the genocide”. The counterpart blog being it is easy to position their view.

Although this blog merits no further discussion, it highlights the often wrong interpretations of the natural selection theories. Eugenics is “just” a political interpretation by some of Darwin’s theorie (see Wikipedia). Darwin himself thought it “absurd to talk of one animal being higher than another” and saw evolution as having no goal.

As a biologist I grew up with the following definition of natural selection.

Natural selection is the process by which favorable heritable traits become more common in successive generations of a population of reproducing organisms, and unfavorable heritable traits become less common, due to differential reproduction of genotypes.

In other words natural selection genetic alterations are mostly random and chance (environment, conditions) will determine whether the genotype exhibiting a new phenotype will continue to exist or even will be more likely to survive (natural selection).

Antibiotic resistance
During my biology study we did all kind of mini-evolution experiments. For instance, we treated bacteria that were deficient for a specific amino-acid (AA) with mutagens and plated them on solid agar plates with or without that particular AA. Only bacteria with a mutation making them independent of that AA would survive on AA-less plates.

Although this is not an experiment of nature, a very similar example of natural selection in action is the development of antibiotic resistance in microorganisms (see wikipedia).


Enhancement of antibiotic resistance by natural selection - modified from wikipedia

Natural populations of bacteria contain considerable variation in their genetic material, primarily as the result of mutations. When exposed to antibiotics, most bacteria die quickly, but some (red in Figure) may have mutations that make them less susceptible. If the exposure to antibiotics is short, these individuals will survive the treatment. This selective elimination of maladapted individuals (lighter colors) from a population is natural selection.

Evolutions within
It is not difficult to see how infectious diseases were driven by natural selection (of the organisms causing these diseases). Because all rules that apply to eukaryotic organisms apply to prokaryotic organisms as well. But I would make a point that evolution and natural selection also takes place at a lower level: that of viruses (non-living organisms, see post about sputnik-virus here) and of “individual cells” within an organism. That is to say: the same mechanisms apply.

Clonal selection and B-cell adaptive immune response.
One example of a cellular evolution is the development of the B cell (and T cell) immune repertoire. B and T cells are cells of
the adaptive immune response. In contrast to the innate immune response, which is always ready to respond to whatever intruder, the adaptive immune response matures throughout life, is antigen-specific and long-living. The specificity of B cells lies in the variable region of their immunoglobulins or antibodies, Y-like molecules, anchored in the B cells’ plasma membrane. There are endless antibody variants and each B cell (and its progeny) produces antibodies with one particular specificity.
How is this diversity established?
In the Pre-B cell phase, when B cells do not produce any immunoglobulins individual gene segments coding for the V, (D) and J regions of the heavy and light chain of the immunoglobulin molecule are randomly assembled to one molecule. The random assembly of 51 V, 27 D and 6 J gene segments provides a minimum of 8.300 different possible combinations for the heavy chain alone, but since the recombination process is not precise and extra nucleotides are inserted the number of possibilities of antibody V region diversity turn out to be greater than that.[2]
(The following excellent animation is recommended: (be sure to choose Open > Antigen Recognition > Recombination)


When an organism encounters a foreign microorganism or other antigen, only those B cells that recognize the antigen are stimulated to divide and to become plasma cells which produce many antibodies specific for the particular antigen. This process is called clonal selection. It results in a B cell repertoire skewed towards the antigens encountered in life. The advantage is that those B cells are selected that have been proved useful. The next time the same antigen is encountered the response is quicker, stronger and more specific, a process called memory.This is also the principle behind vaccination and boostering.

The principle of clonal B cell selection is very similar to the development of antibiotic resistance, discussed above.


Carcinogenesis: Follicular Lymphoma
However, sometimes clones are selected that erroneously react with ‘self’ which results in ‘autoimmunity‘.

Cancer can also be considered as another faulty ‘evolution’, be it within the organism. Cancer cells are better at surviving and reproducing than other cells, because they have escaped the body’s controls. This allows them to increase their population much faster than other cells.

In an interesting editorial, J Breivik comments on the work of Vineis and Berwick[4,5]:

Vineis and Berwick argue that ‘Carcinogenesis, at least for some types of cancer, can be interpreted as the consequence of selection of mutated cells similar to what, in the theory of evolution, occurs at the population level’. Taking a more conclusive stand, I will ague that carcinogenesis is an evolutionary process within the multicellular organism. Evolution by means of natural selection is a scientific principle that reaches far beyond the origin of the species and is applicable to all systems of inheritance, including somatic development.

One example is follicular lymphoma (FL). Follicular lymphoma is characterized by a chromosomal translocation between chromosome 14 and 18, t(14;18), caused by a faulty coupling of the immunoglobulin heavy J chain to the BCL-2 proto-oncogene on chromosome 14 during the normal VDJ-rearrangement process, described above. This mistake leads to a constitutive overexpression of BCL-2, which makes the cell less vulnerable to apoptosis (programmed cell death). Mice bearing a transgene mimicking the BCL-2 translocation have an increased incidence of spontaneous B lymphoid tumors. The lymphomas take many months to develop, however, and the penetrance of disease is low, arguing that BCL-2 overexpression on its own is not highly oncogenic (reviewed in[6]). Indeed our group has shown many years ago that t(14;18) translocations, that were considered specific for follicular lymphoma generally occur in follicular hyperplasias [7] and even in B-cells of healthy individuals [8]. Apparently B cells with the t(14;18) translocation are regularly generated in normal individuals, but only very few cells with the translocation will acquire the additional oncogenic hits necessary to establish the malignant phenotype. Overexpression of BCL-2 only gives the cells a survival advantage. Indeed, according to recent insights [9]:

“Accumulation of genomic alterations and clonal selection account for subsequent progression and transformation. Recently, the role of the immunologic microenvironment of FL in determining clinical behavior and prognosis has been substantiated. Combined genetic and immunologic data may now support a model for the development of FL as a disease of functional B cells in which specific molecular alterations infer intrinsic growth properties of the tumor cells as well as dictate a specific functional cross talk with the immunologic regulatory network resulting in extrinsic growth support.”

The theme of this week inspired me to philosophize about immunity and cancer being examples of evolutionary process. While reading I found that this idea is by no means new; a lot has been written about this concept. For instance in “Understanding Evolution” the writer(s) quite nicely explain the process of evolution within a cell lineage. They first explain that the key elements of the evolutionary process – variation, inheritance, and selective advantage – characterize not just populations of organisms in a particular environment, but also populations of cells within our own bodies.
Furthermore they make the interesting statement that

cancer – even within one person – isn’t a single entity. It’s a diverse and evolving population of cell lineages. A single tumor, for example, is made up of a variety of cell types, produced as the cells proliferated and incurred different mutations. All of this diversity means that the population of cells could easily include a mutant variety that happens to be resistant to any individual chemotherapy drug we might administer. To make matters even more difficult, treating the patient with that drug creates an environment in which the few resistant cancer cells have a strong selective advantage in comparison to other cells. Over time, those resistant cells will increase in frequency and continue to evolve. It’s not surprising then that a simple cure for cancer has yet to be developed: treating even a single type of cancer is a bit like trying to take aim at a whole set of moving targets all at once”

Thus, this challenge helps explain why research has not yet provided us with a cure, but also points the way toward new solutions that take that evolution into account ….


  1. Wikipedia (several pages, as indicated)
  2. Kimball Biology Pages: [A] AgReceptorDiversity (very good background information in dictionary-format)
  3. Evolving Immunity – A Response to Chapter 6 of Darwin’s Black Box. Matt Inlay. [blog] Talkdesign: interesting discussion on whether or not clonal selection system could have evolved in the context of irreducible complexity.
  4. Cancer the evolution-within, by Dan [blogpost] on Migrations (2007/04/18) referring to:
  5. Cancer – evolution within. Breivik, J. Int. J. Epidemiol. (2006) 35, 1161-1162.
  6. The Bcl-2 family: roles in cell survival and oncogenesis. Suzanne Cory1, David C S Huang1 and Jerry M Adam. Oncogene (2003) 22, 8590-8607.
  7. Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. Limpens J, de Jong D, van Krieken JH, Price CG, Young BD, van Ommen GJ, Kluin PM. Oncogene. 1991 Dec;6(12):2271-6.(PubMed-link)(
  8. Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals. Limpens J, Stad R, Vos C, de Vlaam C, de Jong D, van Ommen GJ, Schuuring E, Kluin PM. Blood. 1995 May 1;85(9):2528-36.(PubMed-link)(Google Scholar)
  9. Molecular pathogenesis of follicular lymphoma: a cross talk of genetic and immunologic factors. de Jong D. J Clin Oncol. 2005 Sep 10;23(26):6358-63.(PubMed-link)
  10. Another perspective on cancer: Evolution within. [blog] Understanding Evolution with a detailed description on natural selection within, and the evolution of cancer cells plus possible solutions.


  1. Antibiotic Resistance: wikipedia
  2. Clonal Selection:
  3. Recombination: Evolving Immunity – A Response to Chapter 6 of Darwin’s Black Box, adapted from janeway