The Mouth of the Yellow River Says 是

On October 14, a commenter who identifies himself as "Mouth of the Yellow River" (MOTYR) left one of his usual perceptive remarks in the "For the Saturday Surfer" entry. It concerned a publication by Nobelist Kary Mullis of a "scientific correspondence" in Nature when he was still in graduate school. I was motivated by it to leave a message for MOTYR inviting him to become a contributor to YBYL - in a simple and *extremely* florid Mandarin. (Both can be read  here.)  I ended the comment by expressing the hope that he would say 是 (yes). As the essay below shows, he did. (Otis)

Cancer Genome Sequencing in Wonderland Ad Infinitum

The most extensive results to date concerning the repertoire of point mutations in cancer samples revealed by gene sequencing is out in the 29-author treatise published by one of the science industry’s Holy Trinity (CSN--Cell, Science, Nature) (The Consensus Coding Sequences of Human Breast and Colorectal Cancers, Science 314:268, 2006).  The study costing about $5 million is an analysis that shows that the number of mutations in products of about half of the estimated genes of the human genome from eleven cell  lines or xenografts from breast or colorectal cancers exceeds a mind boggling 800,000+ compared to a normal sample.  Mutations are errors in the four letter alphabet that spells out in words of three the sequence of the 20 amino acid units that make a functional protein. Thousands of functional proteins working together at wide ranges of activity due to thousands of forces that modify them make up a healthy cell. Xenografts are chunks of cancer tissue removed from patients and selected for their ability to survive and grow in the artificial environment under the skin of a mouse without being attacked by the mouse’s immune system so as to provide enough material to analyze.  Cell lines are mixtures of cancer cells selected one step further for their ability to survive and grow in a Petri dish like bacteria independent of their existence in tissue and even the artificial, but more close to physiological environment in the mouse.

Mutations thought to be methodological artifact, sequence differences between the two normal tissue samples, and “silent mutations” that don’t change the amino acid sequence of protein products were subtracted.  [Although each amino acid of a protein is spelled out by the sequence of three letters of a four letter alphabet, the third letter of each triplet for an amino acid can be variable referred to as codon degeneracy.  A mutation to one of the variants that can code for the same amino acid is termed by the authors a “silent mutation”).  It was concluded that a total of 1307 mutations that could affect a protein amino acid sequence occurs in 1149 genes in the samples of the two cancers.

 

When the sequence of the 1149 mutated genes from the cell lines and xenografts was examined in 24 additional tissue samples of tumors taken directly from patients without xenograft or cell culture, an additional 365 mutations in 236 genes was detected showing that different tumor samples have different numbers and types of mutations.  After all this, 921 and 751 mutations of potential interest (MOI’s) in the breast and colorectal tumor samples, respectively, for a total of 1672 cancer MOI’s were noted.  Recall the above numbers need to be doubled since only about half of the suspected coding sequences in the genome were examined.  This is an enormous number of MOI’s to begin to sort through concerning questions of whether any one or combination have anything to do with the individual cancers sampled much less other samples of the same types of tumors.

                                                 [...]

To attempt the impossible task of reducing this mind boggling number of mutations to some sort of meaning in terms of causality, the authors went on to try to rank mutations in terms of genes that might be involved in a tumor property described in the literature in single gene functional studies (CAN-genes for cancer candidate genes) in various tumor models or analyses relative to those that are along for the ride (“passenger mutations”).  Mutations were found in genes that have been previously implicated in diverse cancers and cancer models and others that have not been mentioned before.  Most important was the author’s conclusion that among the 24 breast and colorectal cancer samples examined, the two cancer types exhibit their own signature of mutations and each cancer sample of the same cancer type also exhibits its own signature of CAN-gene mutations and no two individual sample had more than 6 CAN-genes in common.  The grapevine of unpublished results from diverse laboratories like the Sanger Institute in England examining diverse subsets of genes across diverse cancers and samples of the same cancers are reporting in with the same results, the diversity and number of mutations among different cancers is astounding.

The current Science report is being hailed as a landmark study, a “tour de force by other cancer scientists” states Science’s News section (First Pass at Cancer Genome Reveals Complex Landscape, Science 313, 1370, 2006).  Rightly so, this report confirms with the most extensive analysis to date (half of the coding gene sequences in the human genome) what the data from hundreds, thousands of prior studies studying lesser numbers of single genes in diverse cancer-related samples varying from one to a few to hundreds predict despite that the authors interpreted their data as a breakthrough in cause of at least some cancers.  The genome of cancers is a tangled mess awash with mutations, the number and types of mutations vary with the individual type of cancer, an individual cancer within a single type of cancer, the time in progression of an individual cancer within a single type of cancer, how an individual cancer at a specific time in progression within a single type of cancer was sampled (big chunk, little chunk) or manipulated prior to analysis (primary tissue, xenograft or cell line) and location of the individual cancer at a specific time in progression (location within the organ of origin or location outside the organ).  Mutations may be happening at a rate in individual cells within a given cancer faster than the cancer can be sampled and sequenced.  And the mind boggling number and diversity of mutations revealed in this first study among two types of cancers and a few samples of them focus on parts of the total genome that code for proteins.  A proportional number likely occurs all up and down the genome that do not directly have to do with appearance of a protein.  These may be equally important to the properties of cancers as what is seen so far.    

Although the title of the current subject paper is misleading, e.g. “The Consensus Coding Sequences of Breast and Colorectal Cancers,” the report is a landmark because the data in it question whether anything can be learned about cancer, its prevention and treatment from mutational analysis.  It questions the entire single or collective gene mutation theory of cancer causation, at least for what is currently observable as a cancer sample.  It should be the basis for calls for new ideas and re-evaluation of where the basic science, clinical science and pharmaceutical industry are headed in respect to cancer, its understanding and management based on the collective mutation theory of malignancy.  (Continued)

The complete essay is available as a PDF file, here.

 

MOTYR (Mouth of the Yellow  River) is a pen name of a scientist with 30 years experience in basic and applied biomedical research in academic and industrial settings. "By any measure, I have been extremely successful in my chosen profession, continuously supported by the NIH for 27 years. Yet I have never had a single, truly original idea funded when written into one of their grants.”

Comments

[George L. Gabor Miklos]

What can one say...?

The "landmark" Science paper is an almost complete waste of time, and money.

Recall that a very large number of the mutations are changes from CG doublets to TG doublets, which simply indicates deamination of unmethylated Cs to uracil that are then recognized as T in the next replication.

So...in cancer cells the methylation system has gone awry...surprise..surprise.

This sequencing madness won't stop because there are all those machines around...and they need to be fed.

[Peter Duesberg]

At the risk of redundancy, may I say again what I answered when first being asked what I thought of tumor genome sequencing as a way forward.

"Why don't they start with the diploid ones?"

[George]

At the risk of my own redundant addition, permit me to recall the thoughts of the one and only Max Delbruck on the subject of "Homo scientificus", and which served as my introduction to what is now YBYL.

They may be read here.

[Darin C. Brown]

And at the certainty of my own redundancy, and in the spirit with which this essay by MOTYR was obviously written, and in which it has been received by the three "good, *'ole* docs" leaving comments here before me, let me point to some remarks of the mathematician, logician, philosopher, G. Spencer Brown, which serve as the Epilogue to the Serge Lang Archive at the AIDS wiki.

[Roberto Stock]

"But in the last twenty-five years... biochemistry has been made into the handmaiden of the new and ill-defined science of molecular biology of which it is not easy to say in what, if anything, its practitioners are trained thoroughly (...) Those would still accept the jocular definition that I proposed many years ago: Molecular Biology is the practice of biochemistry without a license. (...) This shift has had the consequence that a great number of valid biochemical problems are no longer pursued, since, considered as unfashionable, they are not funded. (...) Twenty morons at the right places can kill a science."

In retrospect: a commentary by Erwin Chargaff on 'Studies on the structure of ribonucleic acids', Bioch. Biophy. Acta 1000: 15-16, 1989.

[Frank]

"There can, however, be little doubt that the whole complex of the natural sciences has become a substitute religion, fulfilling the double roles of mysterious incomprehensibility to the lay public and a means of livelihood for its practitioners. The first function could easily be taken over by another creed or pseudo creed, but not the second. The institutionalization of science as a mass occupation, which began during my lifetime, has brought with it the necessity of its continual growth --- similar in that respect to such mythical entities as the 'gross national product' --- not because there is so much more to discover, but because there are so many who want to be paid to do it. Any attempt at reform is, therefore, met by insincere cries about the 'freedom of scientific inquiry'; and this will be followed by the immediate constitution of all sorts of pressure groups, marching under the banner of Galilei. Entrepreneurs disguised as freedom-fighters may look ludicrous, but they are usually effective, for there is little as irresistible as the momentum of the pocketbook."

Heraclitean Fire by Erwin Chargaff, p.117, 1978.

[Steve]

Peter,

Regarding your diploid comment, and not trying to sound too lazy but I don't want to read all of "Oncogenes and Aneuploidy" right now, are you referring to the fact that there are indeed no diploid cancer genes? (Diploid being the normal state of human genes, if I recall my biology correctly.) Or am I missing something else here?

[Otis]

Steve,

Allow me to answer you on behalf of Prof. Duesberg (although since you apparently know him well enough to call him by his first name, I wonder why you do not ask him personally via telephone or email?) who I do not think has any time to spare to address your "lazy" question.

Be that as it might. Yes. There is no completely clean example of a diploid tumor in the vast medcial literature relating to cancer cytogentics for a very long time.

But you seem to be so consumed by oncogenetics that you even write "diploid cancer genes" instead of diploid cancer cells.

[Steve]

Thanks, Otis. I suppose I should have made a more general comment rather than addressing Prof. Duesberg directly (with whom I am not actually on a first-name basis, but since he signed himself "Peter Duesberg" instead of "Professor Duesberg", I felt that it would be friendlier to refer to him as "Peter").

In any case, I had a feeling that he was trying to be funny and was just double-checking that. I am not in fact "consumed by oncogenetics", just a curious bystander.

[Otis]

Hi Steve,

You are welcome and I hope you forgive my slight over-sensitivity on the point of the salutation to the "near universally despised professor" Duesberg.

And yes, he was being funny, but as is typical, he was also making a very serious point, which I trust you have now gotten.

May I ask you to say either publicly or via email why you read YBYL?

Thanks.

[SE]

Hey, don't know who you are but I did appreciate your very amusing analysis. You really hit the nail on the head on several issues. I must say that being the anti-cancer genome poster boy has taken a bit of a toll on me, especially since the Vogelstein paper. So few people are willing to say what they think it is disheartening and how completely some of them have been bought off is a sight to see. I am especially disappointed in Harold Varmus. Stillman I can understand because he knows nothing about cancer, but Varmus, I thought he had more to him. Anyway, I wish more people would speak up and try to put some rationality in this. I think I am the voice of reason but I am being painted as an old stick in the mud who can't see into the future by people like F. Collins. Such is life, but I really did enjoy your essay. And, of course, I am a big fan of Alice.....

Thanks

S.

[George L. Gabor Miklos]

I have been in correspondence.

I wanted to ensure that I had absolutely and correctly understood the tissue samples used in the Science paper .....so I wrote directly to the authors, and they confirmed my fears.....Oi vey indeed.

People should look very carefully at the samples that were used for sequencing and then compared ....their jaws will drop.

The discovery phase consisted not of DNA from *real primary tissues*...oh no...we couldn't do that!...it consisted of 11 samples of cells that had *metastazied from the colon to the liver*; consisting of *3 cell lines* and *8 xenografts*....human cells grown in mice...now that's an interesting mutational environment. The cell lines were **years old.

For the breast cancer discovery samples there was *no* xenograft derived material; *only* cell line material originally from primary breast tumors...so in this case it was not metastatic material.
For validation, microdissected primary tumor tissue from the breast was used.

Thus the two types of tissue source being compared, liver mets and breast primary differ; one was from metastatic material, the other was from primary tumors! There were also differences as regards cell lines and xenografts....no xenografts were used in the breast samples.

So what we have are metastatic cells being compared to primary tissue.....apples to oranges.

It is also obvious to Blind Freddy...as they say here generically...(and that I wrote before) -- the high frequency of "mutation" CG to TG doublets is likely due to deamination of unmethylated Cs to uracil and their subsequent recognition during replication as Ts. All this tells me is that the cellular methylation controls are whacked around in cancer cells...hello?

Finally the differences in mutation frequencies are meaningless...because of the type of comparisons being made. Different cell types will have different levels of initial methylation...and as those cell types struggle to find their identity in the new unstable aneuploid lineages of the cancer...their methylation characteristics ...and hence their deamination probabilities will differ...hello?

I rest my case Your Honour!

As for Varmus...his nickname is ...

One thing is for sure...deep down I'm not shallow.

your pal on the hill.

People should indeed be encouraged to think outside the box, but only when there is evidence of some thinking going on inside the box.

[SE]

And one more thing..... it is quite possible that many of the repeating mutant genes in cancer, the CAN genes, are mutant not because of a role in cancer, but because they are simply more prone to mutagenesis due to some other common property such as their transcriptional or chromatin state or even the position within the nucleus. A property that would differ between different tissues. I have considered that caveat but until I read your article I did not know how to test that. But you pointed out the solution, to analyze the frequency of synonymous mutations in the same genes. If they are also above average, then the increased frequency of the nonsynonymous mutations would not be significant. Unfortunately the numbers are so small that statistical significance might be a problem. But your essay gave me that idea.

Thanks Mr. Mouth of the Yellow River.