Saturday, November 30, 2013

Great Genomics Book

The book by Dale, Von Schantz and Platt, From Genes to Genomes, is almost perfect. It is a 350 or so page exceptionally well written book describing all the introductory materials one would need to become current with genomes and genomics efforts. As with many of the other books I had around I first looked at this and at a glance set it aside. Then came the moment when I wanted to re-understand something and I opened this book up and I was hooked. It in a clean and clear manner takes the reader from basic DNA principles and through all of the key techniques used in genomic studies today. It avoids getting to complex into any one area and it reads in a straightforward and consistent manner. It is a superb asset for “catching up” and I suspect for first learning the materials.

Chapter 1 is the basic introduction to genes and genomes. It is DNA 101 but it contains little tidbits of essential materials that are all well integrated. One thus starts with a clear understanding where the authors are taking the reader.

Chapter 2 is the material on basic gene cloning. It uses the plasmid approach with bacteriophage and does so without burdening the reader with too much overhead and history. This Chapter discusses technique and technology and the reader is given a logical approach to the basics of cloning. Restriction enzymes are introduced and the material is adequate to have enough depth to see how they can be applied. There are, of course, a lot of implementation questions that are left hanging but that is typical of this study. There is a section on ligation and I would like to have seen this carried over a bit when discussing gene knock-outs. We can understand how the genes ligate but the question is how well does this carry-over the later processes.

Chapter 3 discusses DNA libraries. This is a wonderful summary of the concept. The graphics supplement the text without over powering it. One example of what I call the cook book facts is demonstrated in p 95 when discussing hybridization. Here is the curve showing how as temperature increases the DNA starts to break apart. This is the denaturing of DNA, a concept again used with the PCR analysis. This is less a theoretical or structure issue but one of those cook-book facts that have been added to the tool chest of the Genome builder.

Chapter 4 is the PCR process. Simply it is the separating of DNA, then tagging one end and the other end and going through a temperature sensitive denaturing and rebuilding until what is left is millions of copies of a desired DNA segment. My only complaint here is that the graphic, good, albeit it could be made a bit better with color.

Chapter 5 discusses sequencing and it gives a superb discussion of the Sanger approach. Namely ddNTPs are used with segments and then measured in a gel electrophoresis. I assume that the reader may have had some understanding of the physical details but overall it is clear and exceptionally useful.

The text continues developing other elements used in current day genomics. Chapter 9 is an attempt to provide an overview of microarrays, SNP, and even GWAS and phylogenetics. My problem here is that they are trying to stretch it a bit too far. These are reasonable summaries but to do microarrays justice it may take a bit more detail, and yes color, and the phylogenetics is much too much just a high level summary.

Finally the Glossary is fantastic and worth every page.

The strengths and weakness of the book are simple. On the strength side it covers all the key issues superbly. On the negative side, and this may be perhaps me, I find that almost like Organic Chemistry, in Gene manipulation there still are many cookbook rules that are scattered between the facts and logical constructs. If somehow there could be a clarification of the cook book rule and the well understood logical steps that would be a help.

Overall I would highly recommend this book for almost anyone, from beginner to professional. My focus is clinical and theoretical modelling and analysis, and I have avoided bench work as much as possible. But by reading this book I can see again how much work has been done over the past few decades.

Socrates May Help on This One

As a youth I studied in the Seminary in anticipation of becoming a Franciscan. I had yet to read Ockham nor did I yet have any appreciation for Aquinas, the Dominican.  However my French Christian Brother Teachers would soon let me understand that of all the religious orders that one must always beware of, beware the Jesuits. As I later found out, many Jesuit educated students ended up in the CIA, and one student became President, yes Clinton. John Paul II almost went as far as disbanding the Jesuits as a result of their extreme positions.

In my opinion, the self-assured arrogance of many of these Jesuits has brought forth new forms of “theology”; the kinds of theology we saw foment various revolutions in Latin America[1]. The Jesuits are in my opinion sophists of the first degree, capable of turning a phrase to benefit whatever argument they desire to hold forth.

Thus when we saw the first Jesuit Pope it was no surprise that this may be a bumpy ride, in fact it would make our own current president appear reactionary. Before commenting on the recent note by the current Bishop of Rome let me establish some basic facts.

Catholicism was initially, and had remained for many of the early centuries, a religion based upon the individual. The individual was judged based on what the individual did, not what the group did. Thus the whole concept of Distributed Justice, and here it is worth reading John Ryan, a Catholic Priest in the 19th Century, or as we now seems to call it Social Justice, was a reconstruction of post Constantinian Roman Justice. Namely it was the obligations of a group, and especially a group controlled by the local bishop.

The best example would be to examine Augustine, Bishop of Hippo, who controlled his throng, and their money, through what he knew as the basic principles of Roman law. Gregory I, the Bishop of Rome in the early 7th Century, was before becoming bishop basically the “Mayor” and “Proprietor” of the Roman properties, handed over to the Church, including the very City of Rome. The Church used the denigration of individual wealth as a means of control, a tactic consistent with classic Roman precepts. Furthermore, the post-Constantinian Church used these principles to centralize the “redistribution” into the hands of the local bishops. This allowed the bishop to take the wealth of the few and “redistribute” it in whatever manner they saw fit. It in effect eliminated individual responsibility.

 Christianity was primarily seen in the early Church as duties ascribed to individuals and between individuals. What a person did or did not do was the basis for their redemption. Sacraments were not group exercises, but they were a relationship of the individual with God.The Ten Commandments were individual commandments. The Beatitudes were individual dicta, not what the group should do but what the individual should do. Salvation is not attained via the group, but by singular individual actions. Thus the view that groups, read that Governments, have duties to redistribute wealth, is fundamentally against the principals first ordained.

Charity is not the taking of funds from those who have and then get redistributing the wealth by third parties. Charity is the giving by an individual to others who are in need, and moreover, the helping of those individuals to help themselves and thus in turn to help others. True Charity is helping others succeed as we ourselves may have been fortunate enough to do so. It is an individual and personal obligation. Charity is a bilateral obligation. The giver assist the impoverished, yet the impoverished has a duty to make good, nor just make do, with the gift transferred, thus creating another link in the human chain.

The distortion of this into some third party collective was a Roman artifact, and was not part of the origins of Christianity. Peter Brown has examined and analyzed these transitions from individual to group in Christianity as Christianity was Romanized[2].

Let us consider one other quote[3] by Woodrow Wilson in 1912 before moving on to the Bishop:

We have come upon a very different age from any that preceded us. We have come upon an age when we do not do business in the way i n which we used to do business, when we do not carry on any of the operations of manufacture, sale, transportation, or communication as men used to carry them on. There is a sense in which in our day the individual has been submerged. In most parts of the country men work, not for themselves, not as partners in the old way in which they used to work, but generally as employees,-in a higher or lower grade, of great corporations .

There was a time when corporations played a very minor part in our business affairs, but now they play the chief part, and most men are the servants of the corporations . . . .

Yesterday, and ever since history began, men were related to one another as individuals . . . .

To-day, the everyday relationships of men are largely with great impersonal concerns, with organizations, not with other individual men. Now this is nothing short of a new social age, a new era of human relationships, a new stage-setting for the drama of life.

Now here Wilson is praising the individual and denigrating the corporation. Wilson is almost Jeffersonian in his seeking the old ways and seeing in the new some end point of a fatal state controlled by corporations. Yet it was Wilson who did the most to encumber the individual. Income Tax, the Draft, the War, the oppression of women, again he jailed my grandmother who sought a vote, and Wilson's other Progressive programs of institutionalization and rejection of fundamental individualism.

In reality this period opened up opportunity for all. The Carnegies, Rockefellers, and others, albeit controlling mass wealth for the time, themselves came from little and each in turn demonstrated that it could be done and that in doing so each gave back many fold. Every time I look across from the entrance of Sloan Kettering to Rockefeller I see that long line of giving, individual giving. These people came from nothing. They were not from aristocratic families as was the case in Europe. They demonstrated the ability of the individual to prosper. They were examples for entrepreneurs for decades to come.

The last half of the twentieth century was also a time of individual success and in turn individual giving. The Cornell Weill hospital is the benefactor of not just the named man but of many others, the NYU Langone is also the same donation of an individual. The research conducted in the centers with names on them, and those who were anonymous, are a true sign of that individualism of the entrepreneur. Wilson used the corporations as a means to seek political support and failed to understand the full temporal and social benefits. The U.S., unlike Britain where an aristocracy and Class Society exists, has no class. Anyone may have the chance at the gold ring, and those who get it all too often return it in kind, several fold.

Now let us examine but one paragraph in this recent letter from the current Bishop of Rome. In EVANGELII GAUDIUM the Bishop of Rome states[4]:

54. In this context, some people continue to defend trickle-down theories which assume that economic growth, encouraged by a free market, will inevitably succeed in bringing about greater justice and inclusiveness in the world.

This opinion, which has never been confirmed by the facts, expresses a crude and naïve trust in the goodness of those wielding economic power and in the sacralized workings of the prevailing economic system.

Meanwhile, the excluded are still waiting. To sustain a lifestyle which excludes others, or to sustain enthusiasm for that selfish ideal, a globalization of indifference has developed. Almost without being aware of it, we end up being incapable of feeling compassion at the outcry of the poor, weeping for other people’s pain, and feeling a need to help them, as though all this were someone else’s responsibility and not our own.

The culture of prosperity deadens us; we are thrilled if the market offers us something new to purchase. In the meantime all those lives stunted for lack of opportunity seem a mere spectacle; they fail to move us.

First, as many have already recognized the pejorative of “trickle down” is just that, a wonderful proof of the sophist at work. Only those opposed to free markets and capitalism would use the term. Thus, unlike the many Sophists battled by Socrates, such as in Gorgias and Protagoras, this Sophist comes, from the beginning of his argument, to establish his bona fides.

As Mankiw writes on this as well[5]:

First, throughout history, free-market capitalism has been a great driver of economic growth, and as my colleague Ben Friedman has written, economic growth has been a great driver of a more moral society.

Second, "trickle-down" is not a theory but a pejorative used by those on the left to describe a viewpoint they oppose.  It is equivalent to those on the right referring to the "soak-the-rich" theories of the left.  It is sad to see the pope using a pejorative, rather than encouraging an open-minded discussion of opposing perspectives.

Mankiw is quite observant of the facts. Mankiw understand market capitalism, has examined it in detail, and he has personally participated in the process. Indeed Mankiw is correct in saying that the use of the term is a pejorative, meant as such or not, it reflects a mindset.

But let us examine this paragraph from the current bishop.

1. His criticism of “trickle down” is not based upon any fact or metric but upon some concept, not defined, of justice. This is the classic redistributionist's Distributive Justice model having evolved from the 19th century.

2. There is no assumption about the goodness of those wielding power. There is in Christian dogma the burden placed upon the individual, not the Government, to recognize and need and help remedy it. That means teaching someone how to earn a living, not just feeding them, by being an example for those who need guidance, not just once but for a life time, by seeing a need for money, if that is the case, and helping provide it and the other non-monetary needs as may be required. If one has and denies and denigrates those who are less advantaged, or worse oppresses them, then that is an individual sin. There is no real community sin; it is only an agglomeration of individual errors. Here the Bishop errs.

3. We do not see the cries of the sick, dying and oppressed? The Beatitudes were individual directives, they said we are to visit the sick, help them. If a person is ill, without support, then it is our individual responsibility to “nurse” them, to go out of our way to visit them and bring them from the brink. It is not, from a Christian perspective, for us to be taxed and then the Government hands out money in our stead. Again the Bishop errs.

4. Finally what drives humankind in many cases is the movement forward of civilization, of humanity. Those who work in cancer therapy may have some ego involvement, may be compensated, but in many of not almost all cases they are individual commitments to make mankind better. When one walks down York Avenue in New York one sees what many wealth have contributed to help others, not only short term help caring for the sick, but in establishing long term efforts to relieve the disease that plague mankind. In fact the statement the Bishop makes, “we are thrilled if the market offers us something new to purchase. In the meantime all those lives stunted for lack of opportunity seem a mere spectacle”, reflects a total lack of knowledge what a few wealth individuals have done for millions. Even more so, there are thousands more who have financially given, individually and of their total free will, to that which those with a great deal more have set a foundation for. The Bishop not only errs, but he seems to either be deliberately ignorant of this process or totally denies individual duties as the sole path of rectification.

Individualism is not a concept of individual isolationism. It is a principle of individual responsibility and duty, of the belief that all individuals are equal, have equal opportunities, and that given that opportunity that they can achieve whatever they can perforce of their individual efforts. There is no requirement for redistribution; there is in fact a denial of any Distributive Justice, if each individual has unfettered opportunity and balance.

One must attempt to deconstruct what the Bishop is saying, since he now is saying it for the world, not for those in Argentina.

Perhaps we need to have a Columbanus,  an Irish monk who argued continuously with Gregory I  over issue after issue. The Irish never had the hand of Rome controlling them and thus did not have the fear of Rome. They feared God but not man. Perhaps another such dialog is timely.

[1] See G. Gutierrez, A Theology of Liberation, Orbis (Maryknoll, NY) 1988. This is one of the classic works which John Paul II was so concerned about regarding Liberation Theology, fundamentally a philosophy of Distributed Justice amongst the indigents in South America.

[2] See Peter Brown, Through the Eye of a Needle: Wealth, the Fall of Rome, and the Making of Christianity in the West, 350-550 AD, Princeton University Press, 2012.

[3] See Diner, S., A Very Different Age, Americans of the Progressive Era, Hill and Wang (New York) 1998. The Preface.

Monday, November 25, 2013

Personal Genome

There has been an explosive growth in people wanting to get their genome analyzed. The FDA last Friday issued what in my opinion appears to be almost a cease and desist order, or as they phrase it:

must immediately discontinue marketing the PGS until such time as it receives FDA marketing authorization for the device.

 Specifically the FDA claims:

This product is a device within the meaning of section 201(h) of the FD&C Act, 21 U.S.C. 321(h), because it is intended for use in the diagnosis of disease or other conditions or in the cure, mitigation, treatment, or prevention of disease, or is intended to affect the structure or function of the body. For example, your company’s website at ... markets the PGS for providing “health reports on 254 diseases and conditions,” including categories such as “carrier status,” “health risks,” and “drug response,” and specifically as a “first step in prevention” that enables users to “take steps toward mitigating serious diseases” such as diabetes, coronary heart disease, and breast cancer. Most of the intended uses for PGS listed on your website, a list that has grown over time, are medical device uses under section 201(h) of the FD&C Act. Most of these uses have not been classified and thus require premarket approval or de novo classification, as FDA has explained to you on numerous occasions.

 There always has been a set of concerns here. Most people have no clue what some of these readings mean. Also the reading may be at best reflective a a still questionable result. One should ask even if most physicians have the ability to ascertain the results.

There is also the question of what rights have you lost by sending your DNA to some third party.

This has always been an issue and the FDA may very well have taken a proper and necessary step. It will be interesting to follow this.

Divergent Transcription: An Interesting Tale!

In a recent paper by Wu and Sharp the authors discuss the concept of Divergent Transcription. Simply this is a study of all the transcription that generally goes nowhere but from time to time does go somewhere and in this case the development of new genes.

A few decades ago when we looked at the DNA world we thought of it in terms of the Dogma: DNA to RNA to Proteins. Then the proteins did things.

Then we found that we had about 3 Billion base pairs and only about 20,000 genes. That means that we used only about 1-2% of our bases and the other 98-99% were not really used, but not really. That unused DNA was actually used in bits and pieces. There was a ton on non-coding RNA floating all over.

Thus we might think that decades ago the cell was filled with some well-organized proteins, coming from a well-orchestrated RNA process of translation. It was like an airport in the US, with all the people, base pairs, and lining up with the TSA, the promoters, and moving thorough the scanners in order, each being read for proper ID, the scanner being the RNA polymerase and coming out as ticked passengers grouping at each waiting area for the assigned flight. Each waiting area was the proteins composed of the translated bases now nucleic acids. Organized, controlled, and no unverified interlopers.

But now we look again and it really appears like Penn Station in New York. Doors all over, no lining up, people going on Amtrak, LIRR, NJ Transit, subways, no waiting, no seats, no tickets, no security. Then there are vagrants checking out the trash bins, and dozens of other types just wandering and looking. Order may be there but not the type we see at say Newark. There are, if you will, big RNAs and little RNAs, RNAs destined to become proteins, namely passengers on some transport, but there are also just lots of little segments of RNA going nowhere. These are the equivalent of non-coding RNAs just wandering around, crowding up the floor, slowing down the passengers, and at time changing who goes where.

The Model

Wu and Sharp conclude:

we propose that divergent transcription at promoters and enhancers results in changes of the transcribed DNA sequences that over evolutionary time drive new gene origination in the transcribed regions. Although the models proposed here are consistent with significant available data, systematic tests of these models await further advances such as in-depth characterization of additional genomes and experiments designed to test specific hypothesis. Over evolutionary times, genes formed through divergent transcription can be shuffled to other locations losing their evolutionary context. We envision future studies will uncover more functional surprises from divergent transcription, and illuminate how intergenic transcription is integrated into the cellular transcriptome.

Divergent Transcription is transcription that follows a different path than the organized transcription that we think of in a highly organized structure. As Seila et al stated:

Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site–associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.

As Wu and Sharp recount the classic transcription we use their description:

In the textbook model of a eukaryotic promoter, the directionality is set by the arrangement of an upstream cis-element region followed by a core promoter. The cis-elements are bound by sequence-specific transcription factors, whereas the core promoter is bound by TATA-binding protein (TBP) and other factors that recruit the core transcription machinery.

Most mammalian promoters lack a TATA element (TATA-less) and are CpG rich. For these promoters, TBP is recruited through sequence-specific transcription factors such as Sp1 that bind CpG-rich sequences and components of the TFIID complex that have little sequence specificity.

Thus, in the absence of strong TATA elements such as for CpG island promoters, TBP-complexes are recruited on both sides of the transcription factors to form preinitiation complexes in both orientations.

This model is supported by the observation that divergent transcription occurs at most promoters that are associated with CpG islands in mammals, whereas promoters with TATA elements in mammals and worm are associated with unidirectional transcription

We demonstrate the two concepts below using a modified graphic from Wu and Sharp. We show the TATA binding site on the gene and we show the TBP, the TATA binding protein and a mediator and ultimately the RNA Pol II. This is a classic unidirectional process moving across the exons and generating mRNA which is then cleaned and changed to a protein. The related cDNA do not show any of this underlying complexity.

Now below this is a second process, but now we show both forward and backward transcription. This requires a bi-directional promoter which Wu and Sharp discuss.

Wu and Sharp then argue that the model can be characterized by the system below. We have modified their graphic so that we may take a small step further. The Figure below depicts the four processes they consider:

1. Transcription: This is the classic transcription process of taking DNA and changing it to RNA, usually an mRNA.

2. G+T Content: This is the G and T content of the intron and the propensity for mutations to occur in that area and thus setting up a region for the introduction of new gene type sequences.

3. U1 Process: There are small nuclear RNAs used to splice RNA segments together and these are called spliceosomes. One of them us the U1 snRNA. As the mRNA segments are produced they get spliced together by these nuclear RNA segments. They are powerful elements found in the nucleus.

4. PAS Process: The poly(A) is described as, from Baynes & Dominiczak, pp 430-432, as: At the 3' end of all eukaryotic mRNAs (with the exception of histone mRNAs), a polyadenosine track is added, termed the polyA tail. The adenosine residues are not encoded by the DNA but instead are added by the action of poly(A) polymerase using ATP as a substrate. This polyA tail is frequently >250 nucleotides in length. Although it is still susceptible to the action of exo-RNases, the presence of the polyA tail significantly increases the lifetime of mRNA. The presence of the polyA tail has historically been used to isolate mRNA from eukaryotic cells.

We now combine these elements into the Wu and Sharp dynamic, as modified, below:
 We can then represent this model by the meta-equation below:

Here we represent GT, T, U and PAS as some measure of each of the four processes represented in the diagram. Admittedly this is at best an ad hoc representation but it does demonstrate that indeed we have some form of dynamical system and in turn this system depending on whatever the constants are can become an unstable and ever growing process.

New Genes

Out of this process Wu and Sharp argue that new genes can be born. This is an ingenious and compelling argument. The time scale for such a development is not specified but perhaps it may be intuited. Also actual changes have yet to be fully observed from beginning to end. Yet the pieces are logically consistent and are all supported by the evidence.

First a brief summary of the splicesome (from Baynes and Dominiczak, pp 430-432)

In the more complicated posttranscriptional processing of eukaryotic mRNAs, sequences called introns (intravening sequences) are removed from the primary transcript and the remaining segments, termed exons (expressed sequences), are ligated to form a functional RNA.

This process involves a large complex of proteins and auxiliary RNAs called small nuclear RNAs (snRNAs), which interact to form a spliceosome. The function of the five snRNAs (U1, U2, U4, U5, U6) in the spliceosome is to help position reacting groups within the substrate mRNA molecule, so that the introns can be removed and the appropriate exons can be spliced together precisely. The snRNAs accomplish this task by binding, through base-pairing interactions, with the sites on the mRNA that represent intron/exon boundaries. Accompanying protein factors are responsible for holding the reacting components together to facilitate the reaction.

We summarize the U below:

165 nt
Binds the 5' exon/intron boundary
185 nt
Binds the branch site on the intron
145 nt
Helps assemble the spliceosome
116 nt
Binds the 3' intron/exon boundary
106 nt
Displaces U1 after first rearrangement

We explain how this may work in the Figure below adapted from Wu and Sharp. There is on the left a progression of changes in a segment of DNA which would normally read left to right with the inclusion of a new segment from right to left. The PAS sites, three as shown in the Figure below, are covered by RNA segments ultimately allowing the creation of an Exon and Intron. The process is further elucidated on the right where are gene is putatively relocated from one chromosome to another or even just duplicated.
Let us use Wu and Sharp’s text and go through the argument. They proceed as follows:

One consequence of transcription is that it can cause mutations, especially on the coding (nontranscribed) strand.

During transcription, transient R loops can be formed behind the transcribing RNA polymerase II, exposing the coding strand as single-stranded DNA, whereas the noncoding strand is base paired with and thus protected by the nascent RNA.

The lack of splicing signals in the divergent transcript also makes it more vulnerable to R loop formation, as splicing factors have been implicated in suppressing R loop formation.

 In addition, divergent transcription generates negative supercoiling at promoters, which facilitates DNA unwinding and promotes R loop formation.

As a consequence of R loop formation, the single-stranded coding strand is vulnerable to mutagenic processes, such as cleavage, deamination, and depurination. Genomics studies have shown that during mammalian evolution, transcribed regions accumulate G and T bases on the coding strand, relative to the noncoding strand or nontranscribed regions.

Evidence suggests that such strand bias may result from passive effects of deamination, transcription-coupled repair, and somatic hypermutation pathways in germ cell-transcribed genes, in the absence of selection.

Accumulation of G and T content on the coding strand will strengthen the U1-PAS axis.

A-rich sequences such as PAS (AATAAA) are likely to be lost when the genomic DNA accumulates G and T.

In contrast, G+T-rich sequences, such as U1 snRNP-binding sites (e.g., resembling 50 splice sites, G|GTAAGT and G|GTGAGT), are likely to emerge in these regions. Since promoter-proximal PAS reduces transcriptional activity, the loss of PAS and gain of U1 sites should contribute to lengthening of the transcribed region as well as its more robust transcription.

The gain of U1 sites could also enhance transcription by recruiting basal transcription initiation factors or elongation factors.

Therefore a positive feedback loop is formed: active transcription causes the coding strand to accumulate sequence changes favoring higher transcription activity.

As noted above, strengthening of the U1-PAS axis also favors extension of the transcribed region. Being longer gives the transcript several advantages: by chance longer RNAs are more likely to contain additional splicing signals such as a 30 splice site to become spliced, or binding sites for splicing-independent nuclear export factors, thus escaping nuclear exosome degradation by packaging and exporting to cytoplasm .

Longer RNAs are also more likely to carry an open reading frame, either generated de novo or by incorporation of gene remnants.

 Once in the cytoplasm, the RNA should at some frequency be translated into short polypeptides due to widespread translational activity.

Some of the polypeptides may provide advantage to the organism and become fixed in the population, thereby forming a new gene.

Thus we have seen a mechanism for new gene creation and insertion.


These are a very powerful set if insights and observations. They have significant conclusions as has been articulated by those in Sharp’s Lab. The metaphor of a train station with wandering fragments of often “useless” RNA has certain merit. However all too often those fragments are not useless but have ways of interfering and disrupting the normal progress of cellular dynamics.

We now pose a few observation which may have some merit.

1. Somatic vs Germline: These changes seem to be mitotic in nature and thus are reflected in somatic cells. What is the impact in meiosis and germ line cells? Namely can these mutations be carried forward and be selected out in subsequent generations? Or is this process one almost exclusively found in somatic cells and thus may be causes for such diseases as the cancers? I could not find a clear path to follow here.

2. Causation: What causes some of these processes. Many if not most of the links are presented and explained but ultimate causality is missing.

3. Frequency: How frequently do these changes occur? Are they rare or common and at what rate do they occur?  What are the overall temporal dynamics of these processes. Can we examine genomes and ascertain where they might occur. We all too often just skip over the Introns, focusing on the Exons and their resultant expression. There also are many regions of the Exons that are not expressed, and are they part of this phenomenon as well?

4. Reaction Dynamics: The actual reaction dynamics could possibly be explained and modelled. We have presented a meta model solely for the visualization of what may happen. It is expected that the model is most likely non-linear and more complex. In fact the actual metrics being measured and modelled are still in question. However not withstanding that we can envision a dynamic model exhibiting not only stability issues but also oscilliatory effects.

5. Methylation and Epigenetic Factors: Clearly the CpG islands play an important factor. Methylation has become a significant area of study over the past decade and the processes described herein rely on many of these CpG islands as well. Is methylation a competing process, an allied process, a controlling or mediating process?

6. What are all these RNA fragments doing?: Ultimately we find that a cell may have not only well understood Dogma based proteins and pathways but also a mass of disconnected non coding RNA spinning about in the nucleus and throughout the cell. Thus we ask; what do these snippets do? Are they just wanderers going nowhere and possibly just bumping into those going somewhere or are the truly entities which have predictable effects on pathways? Are they noise or an aberrant signal?

This is a very compelling paper and it presents in an elegant manner the results of the efforts to date. This effort demands to be followed and examined in detail as it progresses.


Baynes   Dominiczak: Medical Biochemistry 3E, Mosby (New York) 2013.
Seila, A., et al, Divergent Transcription from Active Promoters, Science VOL 322 19 December 2008 1849.
Wu, X., P. Sharp, Divergent Transcription: A Driving Force for New Gene Origination? Cell 155, November 21, 2013.

Saturday, November 23, 2013

There Seems to Be a Logical Issue Here

In a recent NY Times piece by one of the Harvard economists, he states as follows:

Another clue to what’s happening in the labor market is the vacancy rate. Although less widely followed than unemployment figures, this rate is its mirror image. To compile the unemployment rate, the Bureau of Labor Statistics surveys households to find workers without jobs. To compile the vacancy rate, the bureau surveys employers to identify jobs without workers. In short, the vacancy rate measures the percentage of available jobs that are currently unfilled. 

Not surprisingly, the vacancy rate is highly cyclical. In recessions, when customers are hard to find, businesses post fewer new jobs. In addition, because the number of job seekers expands, the posted openings are filled quickly. As a result, the vacancy rate falls. Conversely, when the economy recovers, businesses start posting new openings, and jobs are harder to fill, so the vacancy rate rises. 

The recent recession is a case in point. Seven years ago, the vacancy rate was a bit over 3 percent. It fell to a low of 1.6 percent in July 2009, a month after the official trough of the recession. The most recent reading puts it at 2.8 percent. So according to this measure of labor-market tightness, the economy is almost back to normal. 

 Let us examine the statements and numbers and let us see what may be wrong with this logic.

1. We know that the percent of the population employed has dropped precipitously. We have been noting that for five years now. Others seemed to ignore it for the first three years but since I am not an economist I deal with the facts, all the facts.

2. The percent unemployed is a floating and relative number. It helps get to the real number if and only if you know what the real number should be. To me it should be the percent of the population employed before the Recession adjusted for the increase in population.

3. Now the vacancy rate means nothing more than what industry demands. It is a meaningless number and reflective more of productivity than the ability of the economy at large to absorb workers. We may have a 20% vacancy rate if we had massive productivity gains and got rid of 40% of those employed and we may never fill it if the demand is for people who can do real things. Like engineers, electricians, even plumbers, but not fine art majors and not economists.

4. Thus to make any nexus between the total percent of the population employed, the employment vacancy rate, and the economy is specious at best. With the massive improvements in productivity we see less people required to be employed and those in demand have talents which are also in short supply. The work force is NOT homogeneous as one would assume if one were an old fashion economist who viewed the world of workers as factory types.

Thus the argument that we are improving because of an increased vacancy rate is at best wishful thinking. The percent of the total population is increasing, as we have noted, but barely. The economy is much more complex than as stated.

Independent Scotland

The BBC reports on an independent Scotland by March 2016. They state:

Scotland could be independent on 24 March, 2016, if voters back leaving the UK in the independence referendum, the Deputy First Minister has announced.

The date is included in the Scottish government's White Paper, described as a "blueprint" for independence.

Despite that a Catholic still cannot be the Prime Minister and Catholics still have limited rights in the Occupied Counties. They continue:

March 24 is also the anniversary of the Union of the Crowns in 1603. 

A Scotland Office spokesman said: "Naming the date of independence ahead of a referendum result would only weaken the Scottish government's negotiating position if Scotland voted to leave the UK. 

"People in Scotland still don't know the full terms the Scottish government would try to negotiate but the 28 members of the EU, Nato and the rest of the UK would all know that for the Scottish government the date is more important than the deal.

But Ireland has been dominated by the English since the Pope used the fraudulent Donation of Constantine to allow Henry II to donate it to his less than balanced son John.Good luck to the Scots!

Sister Rosita and the MOOCs

In the Fifth Grade I had Sister Rosita for my teacher. Even though I was performing well, somehow I never got the spelling stuff quite right. My readers can amuse themselves reading the blogs wherein I have the right spelling but the wrong word. That is, spell check, not me, I just spell the right word wrong, not the wrong word right.

But what, you may ask; does this anecdote have to do with MOOcs? Simply, MOOCs use computers to grade. Computers do not offer partial credit. You are right or wrong. Over the years I mastered multiple choice tests, Board exams, and the like, not that they prove anything, they are required, but one learns a technique, not the reality of what one started learning in the first place.

But back to MOOCs. I have been taking Lander’s MIT Biology, brilliant course, I know most of it but it is always good to gain a new perspective from one who really knows what they are doing. It is a typical MIT course in many ways, except it is a MOOC.

To explain, allow me again to digress back some almost fifty years when I was teaching at MIT.  As a faculty member when we sat to do the final grades, say in 6.02, the required Electronics course, we had all the Teaching Assistants, Section Instructors, and myself, gather and look at each student. If the student got say a 95 average on all 12 Problem Sets but totally blew the Mid Terms we asked what we did wrong. Clearly the student knew something and each of us knew something of that student. We examined the exam. Frequently we could see that we had a set of problems that if one concept was misinterpreted then all that followed, albeit properly done, rely on a correct first answer and thus was wrong. So what did we do, partial credit, regarded, and alas, the Final and Mid Terms went up. The person came out with the A.

But this is not at all possible with a MOOC. Now I am not after a grade. I have no idea what I would do with one. I am there to learn the material and the process.

Now back to Sister Rosita. Decades later I found that the problem was not my spelling but my particular kind of dyslexia. I can now look forward in the family and see it there and I guess if I could look backward I would see it also. But I can readily understand and inter-relate complex patterns, but somehow have a heck of a time differentiating “a” and “c” and “b” and “d”. But wait, I find that I have an even more challenging time with A, T, G, C! I get the idea but ask me to look at a sequence of five and then go and find the same five on another sheet of paper! Then present say 100 or so bases randomly presented and ask to find say six of a particular pattern, no way, ever. After all is that not why God created computers.

Now back to the MOOC. You see, in one of Lander’s Exams the TAs made you read the gene and you could not readily cut and paste it. I did finally remember how to do it but after the exam. Thus since this was the first part of some 65 point single question, miss that and you were sunk. No partial credit etc.

Now I did not worry but in examining the comments, I never comment on these things, but many students had the same if not similar problem. Thus what is the lesson MOOCs could learn?

Simply, not all students are the same. We all find ways to work around our handicaps, whatever they may be. But when administrators of sorts decide upon “rules” they believe to be correct, then it is incumbent upon them to understand the consequences. MOOCs will never look at every student the way we did half a century ago. Yet they can come close. They can and must become capable of understanding what a student is doing wrong, not just marking it wrong. One learns through one’s mistakes. One learns how to manage “blind spots”. Artificial walls should not be constructed because of the arrogance of some intermediary.

Is Diogenes Still Searchng?

Is Diogenes still looking for truth? Does truth truly exist? Let's try truth in Health Care and especially the progress of the ACA.

According to the WaPo:

By mid-November, the 14 state-based marketplaces reported data showing enrollment has nearly doubled from last month, jumping to about 150,000 from 79,000, according to state and federal statistics. The nonprofit Commonwealth Fund, which has been tracking the data, called the most recent numbers “a November enrollment surge.”

But one must look at the source of this good news. The source, the Fund so named above, is headed by one of the ACA principals, and brother of a Democrat Senator, and manager of the deployment of the EHRs that have somewhat in my opinion made chaos of every medical practice.

The question is; why is the Government not giving out the data? The answer may very lie with the fact they seem to have the tendency to distort it anyway, just look at the employment data. One wonders, in my opinion, why one would ever believe these politically well connected entities.

Truth has taken a real beating lately.

Friday, November 22, 2013

PCa, Oncosomes and Blood Borne Markers

Almost daily one can see new proposed markers for Prostate cancer and its potential metastatic growth. There simply are often too many to have true prognostic value. The major concern that we have expressed over the past few years is the lack of causative structure behind many of these proposed markers. That is, the marker may be reflective of some more complex causative process and it is often that process, albeit unknown or not expressed, that we are seeking so that one may have the ability to develop targeted therapeutics against that target. For example we understand PSA as a marker reflective of an enlarging number of prostate cells but the driver may be any one of several things other than cancer.
(NOTE: The above is from Grasso et al. It is a powerful and current update of genetic profiles of PCa)

In this note we examine two specific areas. The first is recent reporting on exosomes, called Oncosomes in the discussed work. This work covered in a recent and several extensive previous papers examine encapsulated blood borne markers for aggressive PCa. In contrast we examine a second recent result, also a blood borne marker of aggressive behavior, an mRNA of a quite large gene. The second is another blood borne marker, an mRNA from a quite large gene, NAALADL2 or N-acetylated alpha-linked acidic dipeptidase-like 2, located at 3q26.31, a large gene with hundreds of introns[1].

An excellent paper by Grasso et al has discussed the genetic landscape of androgen resistant PCa[2]. The authors summarize their work as follows: 

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. 

Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factorFOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutatedFOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.

The Table that they present is quite useful in this study. The measures are primarily mutational in nature.

We have previously reported on the development of exosomes, small encapsulated particle ejected from cells, often carrying within them marker proteins reflective of the status of an organ. Exosomes have been used for first, a word on terminology. As Simpson and Mathivanan state: 

The first problem relates to the terminologies used in naming eMVs. In the past, isolated eMVs were named based on the sample source from which they were derived. For example, exosomes isolated from dendritic cells were named dexosomes, while cancer cell derived exosomes were referred to as texosomes/oncosomes and prostate cancer cell derived exosomes as prostasomes. This sample material based vesicle naming customization has lead (sic) to different nomenclatures such as epididimosomes, argosomes, exosome-like vesicles, apoptotic blebs, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, exosomes, microparticles, nanoparticles, microvesicles, shedding microvesicles, ectosomes, archeosomes and oncosomes.

Note that a bleb is an outward protrusion from a cell. Recent work by Funkhouser et al has some interesting detailed analyses of this phenomenon.

Let us begin by considering the Oncosome results. We use the term oncosome rather than the more encompassing exosome because that is what is done in the paper.

In that paper the authors make claims as regards to these “oncosomes” and they state the following[3]: 

Prostate cancer cells release atypically large extracellular vesicles (EVs), termed large oncosomes, which may play a role in the tumor microenvironment by transporting bioactive molecules across tissue spaces and through the blood stream. In this study, we applied a novel method for selective isolation of large oncosomes applicable to human platelet-poor plasma, where the presence of caveolin-1-positive large oncosomes identified patients with metastatic disease. This procedure was also used to validate results of a miRNA array performed on heterogeneous populations of EVs isolated from tumorigenic RWPE-2 prostate cells and from isogenic non-tumorigenic RWPE-1 cells. The results showed that distinct classes of miRNAs are expressed at higher levels in EVs derived from the tumorigenic cells in comparison to their non-tumorigenic counterpart. 

Large oncosomes enhanced migration of cancer-associated fibroblasts (CAFs), an effect that was increased by miR-1227, a miRNA abundant in large oncosomes produced by RWPE-2 cells. Our findings suggest that large oncosomes in the circulation report metastatic disease in patients with prostate cancer, and that this class of EV harbors functional molecules that may play a role in conditioning the tumor microenvironment.

Thus the description could best use the general term exosome but we shall remain with and use oncosome throughout. Before continuing it is worth examining some prior research in this area as well. In the 2009 paper the authors state:  

Oncosomes are recently discovered membranous microvesicles that have been implicated in rapid intercellular transfer of oncogenic information from glioblastoma to indolent glioma cells. Although this process resembles paracrine signaling, it involves intercellular transfer of a membrane-bound micro-organelle rather than a soluble protein such as a growth factor or cytokine. In the present study, we show that PCa cells shed membrane-bound vesicles in response to signal transducers. These structures are fairly large (0.5 to f5 Am), originate from nonapoptotic blebs in response to signaling cues, and have biological activity in their free-floating state. We also identify the formin homology protein, DRF3/Dia2, as a protein that seems to functionally inhibit oncosome formation. We also provide the first evidence that chromosomal loss at the DRF3 locus (DIAPH3) is associated with metastatic PCa.

Note that DIAPH3, diaphanous-related formin 3, is a protein that “this gene encodes a member of the diaphanous subfamily of the formin family. Members of this family are involved in actin remodeling and regulate cell movement and adhesion. Mutations in this gene are associated with autosomal dominant auditory neuropathy. Multiple transcript variants encoding different isoforms have been found for this gene[4].  The authors continue with a discussion of DRF3[5] an actin facilitator gene: 

Our data indicate that the actin nucleator DRF3 is capable of inhibiting oncosome formation, because DRF3 knockdown by RNAi increased blebbing in DU145 cells, particularly in the presence of EGF. DRF3is expressed by LNCaP, DU145, and PC-3 human prostate cell lines. Formin homology proteins mediate cytoskeletal dynamics and, as a group, have been implicated in a wide range of cellular functions, including motility and vesicular trafficking. The formin FHOD1, which exhibits 45% sequence homology to DRF3, was recently implicated in Src-dependent plasma membrane blebbing.

It should be recalled that blebs are cell protrusions due to cytoskeleton breakdown and blebbing is the process of creating such nascent vesicles. The authors continue:

Human DRF3 is not well-studied, although analyses of the mouse homologue Drf3, and the close mouse paralog, Drf1/mDia1, indicate that DRF3likely mediates actin filament nucleation and elongation and microtubule stability Our experiments suggest that oncosome transfer between tumor cells, or between tumor and stroma, could play a role in propagation of aggressive behavior within the tumor microenvironment…… oncosome exchange is markedly different from paracrine effects induced by soluble ligands. However, this process could result in amplification of paracrine pathways through intercellular sharing of membrane-associated signaling complexes. Although our study focuses on PCa, a similar microvesicular transfer mechanism may operate in other tumor systems.

From the 2012 paper we have the following diagram which has been modified and simplified. The description is one of a tumor mass eluding encapsulated exosomes in which are specific proteins. It could possibly be equally likely that the exosomes may encapsulate mRNA as well which is a putative marker. This description also shows that any localization of the source is in question.

The authors conclude in that paper: 

Quantitation of circulating tumor cells (CTCs) is being evaluated to assess the risk of disease progression for prostate and other types of cancer. However, the clinical significance of CTCs remains to be established because of their extremely small number in peripheral blood compared with the number of blood cells. We have demonstrated that large oncosome-like structures can be separated from plasma in a manner that does not require the capture of CTCs or other cells. The molecular characterization of large oncosomes may potentially offer a more sensitive and specific liquid biopsy than CTCs for patient selection, monitoring of treatment efficacy, and assessment of drug resistance.

There seems to be a significant interest in various forms of circulating tumor cells, exosomes or otherwise. In a previous note we observed work done on extracting exosomes from urine as a way to assess PCa[6]. This approach was putatively a localized approach.

The observations of the press are oftentimes of use in understanding how these results are interpreted. Several comments are below[7]: 

Investigators in the Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute have made extensive progress in understanding the molecular mechanisms of disease progression. These results may help scientists better understand the prognosis of patients diagnosed with advanced prostate cancer. …"One of the long-standing difficulties in treating men with advanced prostate cancer has been predicting the response to given therapies or treatments," said Di Vizio, associate professor in the Department of Surgery, Department of Pathology and Laboratory Medicine and Department of Biomedical Sciences. "These latest research findings provide tangible insight into the molecular and structural phenomena that result in prostate cancer metastases. They have the potential to create a new source of biomarkers and an innovative standard of care. These findings may also help distinguish individualized treatment plans best suited for each patient." The new source of biomarkers is large oncosomes, which are vesicles released from aggressive prostate cancer cells with highly migratory features. These large oncosomes carry tumor molecules and have been shown in previous studies to contribute to tumor progression. This study demonstrates, for the first time in human samples, that identification of circulating large oncosomes can be an indicator of patients with more aggressive, treatment-resistant disease. Also notable, researchers found that large oncosomes contain microRNA, a molecule that regulates several biological processes now proven to influence tumor progression.

We now briefly examine another recent market for metastatic behavior. Specifically we discuss the NAALADL2 gene. The work of Whitaker et al has received some recent interest because it alleges to allow, upon biopsy, to ascertain if the lesion was of an aggressive nature. The authors also argue that this measure can be ascertained vial circulating mRNA related to the gene as well. Whether this circulating mRNA is exosome encapsulated is open for discussion.

From the paper by Whitaker et al[8]:

N-acetyl-L-aspartyl-L-glutamate peptidase-like 2 (NAALADL2) is a member of the glutamate carboxypeptidase II family, best characterized by prostate-specific membrane antigen (PSMA/NAALAD1). Using immunohistochemistry (IHC), we have shown overexpression of NAALADL2 in colon and prostate tumours when compared with benign tissue. In prostate cancer, NAALADL2 expression was associated with stage and Grade, as well as circulating mRNA levels of the NAALADL2 gene. Overexpression of NAALADL2 was shown to predict poor survival following radical prostatectomy. In contrast to PSMA/NAALAD1, NAALADL2 was localized at the basal cell surface where it promotes adhesion to extracellular matrix proteins. Using stable knockdown and overexpression cell lines, we have demonstrated NAALADL2-dependent changes in cell migration, invasion and colony-forming potential. Expression arrays of the knockdown and overexpression cell lines have identified nine genes that co-expressed with NAALADL2, which included membrane proteins and genes known to be androgen regulated, including the prostate cancer biomarkers AGR2 and SPON2. Androgen regulation was confirmed in a number of these genes, although NAALADL2 itself was not found to be androgen regulated. 

NAALADL2 was also found to regulate levels of Ser133 phosphorylated C-AMP-binding protein (CREB), a master regulator of a number of cellular processes involved in cancer development and progression. In combination, these data suggest that changes in expression of NAALADL2 can impact upon a number of pro-oncogenic pathways and processes, making it a useful biomarker for both diagnosis and prognosis.

Unfortunately the complete pathway models for NAALDL2 do not appear to be fully known. On the one hand the authors argue that it is androgen receptor independent, but that it plays a role in:

1.     Cellular adhesion
2.     Cell migration
3.     Colony forming potential
4.     Invasion

In the UK, this result has received considerable attention. For example, from the NHS report[9]: 

"Prostate cancer patients could be screened to detect aggressive tumours after scientists identified a protein linked to severe forms of the disease," reports The Daily Telegraph. The news is based on the results of a complex laboratory study looking at a protein called NAALADL2. Scientists found that levels of NAALADL2 were high in prostate cancer when compared with healthy tissue, and levels were higher in more aggressive and more extensive prostate tumours. The level of the protein found in the tumours was also linked to whether men survived without recurrence of the cancer and overall survival after having radical prostatectomies (surgery to remove prostate cancer). This is exciting news as one of the biggest problems in helping men with prostate cancer is estimating the likely outcome. Some prostate cancers cause no or few symptoms and do not have any impact on life expectancy – doctors may tell you that "many men die with prostate cancer, not of prostate cancer". Other prostate cancers can be extremely aggressive. Around 10,000 men die of the disease in the UK per year. A test that could accurately identify high-risk cancers could potentially save lives and spare men with low-risk cancers unnecessary testing and treatment. So far this is early-stage research. The next hurdle is to see if the results of the lab research can be applied in the real world, and, most importantly, whether it can be used to help improve outcomes for men with prostate cancer. The researchers initially looked to see if the NAALADL2 protein was present in a range of normal and tumour tissues from different parts of the body.

The report, also in PubMed, then reports on the specific results regarding aggressive PCa. They state (as modified): 

They then looked at whether the presence of NAALADL2 could differentiate between benign and cancerous tissue, and whether it could predict survival. Prostate tissue was taken from men who had radical prostatectomies (surgery to remove the prostate cancer) in Cambridge or Stockholm. The researchers then investigated the localisation of NAALADL2 within the cell, what cells making NAALADL2 can do, and which other genes are switched on (expressed) in combination with NAALADL2. What were the basic results? NAALADL2 was present at high levels in colon and prostate cancers.  By measuring the amount of the protein, researchers were able to distinguish between benign and cancerous prostate tissue with a relatively good level of accuracy. They found that in a group of samples from men in Cambridge:The level of sensitivity was 86% (sensitivity is the percentage of cancerous samples that were correctly given a positive result) , The level of specificity was also 86% (specificity is the percentage of benign samples correctly given a negative result) , Similar findings were seen in samples from a group of men from Stockholm.

Although this is a good value for sensitivity and specificity it is only for a small sample and only for ascertaining benign vs malignant. Aggressiveness may very well be another issue. They also continue as follows: 

Levels of NAALADL2 protein increased with the increasing aggressiveness of the prostate cancer, based on the microscopic appearance of the tissue (Gleason grade). Levels of NAALADL2 protein also increased with cancer stage (the extent and spread of the tumour), particularly between T2 (cancer confined to the prostate gland) and T3 (cancer that has begun to grow and spread outside the prostate into the seminal vesicles, the glands that produce the fluid component of semen). Levels of NAALADL2 RNA (mRNA of NAALADL2 in the blood stream) in the blood were found to be higher in men with biopsy-confirmed prostate cancer, compared with men who had raised prostate specific antigen (another protein associated with prostate cancer) but a negative biopsy. 

One of the concerns one would have here is that it is known that biopsies are often non-conclusive and require multiple tries. This is especially true with the older methodologies of sextant cores. With high density cores, 24 and higher, and with repeat biopsies at say 9 months, one can reduce but not eliminate this risk. Secondly, there is the issue of how the mRNA is obtained from the blood. We have been discussing exosomes but one suspects that the UK approach is not that but actual mRNA extraction. Blood extraction can be problematic because one does not know from whence it came. It could be prostate originated thus a local but aggressive disease or it could already have metastasized, having established itself in the bone. This is one of the major difficulties of blood borne markers. 

The researchers then looked at whether levels of NAALADL2 protein could predict survival. One hundred and four men had radical prostatectomies in Cambridge, and 38 had recurrence of the cancer over a median follow-up period of 86 months.There was a trend that higher levels of NAALADL2 led to poorer outcomes, but this wasn't statistically significant. The researchers suggested that this might be because of the small number of men: the smaller the sample size, the less "statistical power" the results have.  They then looked at data from Stockholm: in this cohort, there were 252 men, and 101 of them had recurrence over a median follow-up of 61 months.  Of men with low levels of NAALADL2, 79.9% had no relapse at five years. Five-year recurrence-free survival was reduced to 72.5% for men with moderate levels of protein, and 65.3% for men with high levels of protein (hazard ratio 1.9). The result was still significant after adjusting for a number of factors, including the Gleason grade and cancer stage. Levels of NAALADL2 could also predict poor survival in low-risk patients (patients with low Gleason grades and cancer stage). Five-year survival was 93% in men with low levels of NAALADL2 and 45% in men with high levels of NAALADL2. The researchers found that NAALADL2 protein on the basal (base) cell surface, where it promotes cell adhesion, migration (movement) and invasion (movement into tissue). They suggest that this could allow cells to escape the prostatic capsule and form tumours elsewhere. NAALADL2 was found to be expressed alongside androgen-related genes and prostate cancer biomarkers. How did the researchers interpret the results? The researchers conclude that, "NAALADL2 protein is expressed in a number of cancers, and highly expressed in prostate cancer, where it predicts for relapse following radical prostatectomy". They go on to say that, "These data suggest that changes in expression of NAALADL2 can impact upon a number of pathways [involved in cancer development], making it a useful biomarker for both diagnosis and prognosis."

The size of the samples is exceedingly small and thus although this result is compelling and of interest it is clearly not of clinical significance. Thousands of patients would have to be examined and one of the most confounding elements would be the type of biopsies and the way in which they were performed.

Now the work in this area has also led to a patent filing and issuance.  From the Patent[10]: 

The invention features methods for detecting prostate cancer, especially hormone-refractory prostate cancer (HRPC) or castration-resistant prostate cancer (CRPC), by detecting over-expression of PKIB or NAALADL2 compared the normal organs. Also disclosed are methods of identifying compounds for treating and preventing prostate cancer including HRPC, based on the over-expression of PKIB or NAALADL2 in the prostate cancer, the cell proliferation function of PKIB or NAALADL2, the intracellular localization of PKIB or NAALADL2 or the interaction between PKIB and PKA-C. Also, provided are a method for treating prostate cancer by administering a double-stranded molecule against the PKIB or NAALADL2 gene. The invention also provides products, including the double-stranded molecules and vectors encoding them, as well as compositions comprising the molecules or vectors, useful in the provided methods. An isolated double-stranded molecule, which when introduced into a cell, inhibits in vivo expression of PKIB or NAALADL2 and cell proliferation, which double stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule.

It is clear that the researchers have staked out their territory. But also have many others who have patented various other markers.

As one reads the current literature and discusses the issue of identifying what PCa is aggressive the general consensus is still that the problem has no simple solution. These two recent attempts identify new markers which may be useful. Blood borne markers, mRNAs or exosomes, have some improved predictive value in some limited trials. However there is not as of yet a true statistically significant set of such markers which we can rely upon.

The problem quite simply is as follows. The US Task Force has recently stated that there should be no PSA testing because most PCa are indolent. As we have argued again and again, the problem is that most is not all and that for that 5-10% who has this aggressive form that such a recommendation is most likely a death sentence.

The work in these two areas demonstrates two things. First, the aggressive form is quite prevalent and is indeed aggressive. Second that there are many putative markers, blood borne, which can provide reasonably determinations as to the aggressiveness.

Thus the USPTF recommendations are in our opinion without merit since the deny the severity and prevalence of the aggressive PCa status. The work herein is of significant merit and should be closely followed as one gets a better understanding of this disease.

There are however several issues which need clarification. They are:

1. Pathway Implications: As we have argued extensively before, understanding the pathway interactions is essential. For example, what does the NAALADL2 gene do and how specifically does it do what it does. This would lead us to understand whether this is an issue of that gene itself or of some promoter of that gene, and if so what promoter. The genetic network is essential to be a part of diagnosis, prognosis and therapeutics.

2. Stem Cell Issues: As with so many issues regarding PCa there is always the issue of a stem cell. Is the stem cell the one forcing the overproduction? The questions are quite substantial once we address this area.

3. Localization: Blood borne markers have significance but the question is from where these molecules originate. The concern we have expressed internally is that they may be early mets and not the localized tumor. To some degree that is the advantage of the urine based test, it provides a modicum of localization.

4. Statistical Significance: These tests are on small samples. One needs larger scale trials to determine the proper prognostic result. However with all of the putative markers available one could expect to see this being done in a parallel trial. The reason is because perhaps there are linkages which may come about in such a trial. We believe that single thread trials may fail to bring forth the power of multiple markers.

1.               DiVizio, D., et al, Large Oncosomes in Human Prostate Cancer Tissues and in the Circulation of Mice with Metastatic Disease,  The American Journal of Pathology, Vol. 181, No. 5, November 2012.
2.               DiVizio, D., et al, Oncosome Formation in Prostate Cancer: Association with a Region of Frequent Chromosomal Deletion in Metastatic Disease, Cancer Res 2009; 69: (13). July 1, 2009.
3.               Funkhouser, et al, Mechanical model of blebbing in nuclear lamin meshworks,
4.               Grasso, C., et al, The mutational landscape of lethal castration-resistant prostate cancer, Nature, July, 2012.
7.               Morello, M., et al, Large oncosomes mediate intercellular transfer of functional microRNA, Cell Cycle V 12 N 22 2013.
8.               R. Simpson, R., S Mathivanan, Extracellular Microvesicles: The Need for Internationally Recognized Nomenclature and Stringent Purification Criteria, J Proteomics Bioinform 2012, 5:2.
9.               Whitaker, H., et al, N-acetyl-L-aspartyl-L-glutamate peptidase-like 2 is overexpressed in cancer and promotes a pro-migratory and pro-metastatic phenotype, Oncogene, (18 November 2013).

[3] Large oncosomes mediate intercellular transfer of functional microRNA, Matteo Morello, Valentina R Minciacchi, Paola de Candia, Julie Yang, Edwin Posadas, Hyung Kim, Duncan Griffiths, Neil Bhowmick, Leland WK Chung, Paolo Gandellini, Michael R Freeman, Francesca Demichelis, Dolores Di Vizio, Cell Cycle V 12 N 22 2013. also see