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A Close Look at a Cancer Genome

Ever since gene sequencing became feasible (for several values of “feasible”!) it’s been of great interest to look at the genetic material of cancerous cells. It’s been clear from very early on that there are many changes, mutations, rearrangements, shifts, etc. in a cancer cell’s DNA, and it’s been equally clear that these do not tell a simple or clear-cut story. Genomic instability itself is a key feature of many tumor lines, and genomic instability comes in a lot of varieties.

Here’s a new paper that illustrated that in even more detail than we’ve had before. The authors are looking at a cell line that’s considered a model of HER2+ breast cancer, SK-BR-3. And they’re examining it via a (relatively) newer method, long-read sequencing. That’s been coming on for some years now, and this is just the sort of paper that illustrates how it can be valuable. As many readers will know, many current DNA sequencing methods work by breaking genomic DNA down into shorter fragments and reading those, then computationally assembling the original sequence from all the various breaks and overlaps. (This, indeed, was one of the key innovations during the Human Genome Project, and its development and eventual acceptance make for quite a tale). Current short-read sequencers work with roughly a few hundred base pairs at a time, but long-read sequencers up that to several thousand. That lets you get a better look at variants and rearrangements that get missed or averaged out when you chop things up too finely.

Now, short-read “shotgun sequencing” was a real breakthrough – the human genome would not have been unveiled at that famous press conference at anywhere near that date without it. But even as everyone was taking bows, waving to the press, or perhaps exchanging poisonous glances with their collaborators, the question was hanging in the air: what does it mean to have “sequenced the human genome”? Everyone’s genome is different. To some extent, that announcement was, I’m pretty sure, that we’d at least partially sequenced Craig Venter. But Craig Venter ≠ humanity at large (a statement which will come as a relief to no small number of people).

Looking at genomic DNA is like looking at the night sky. Those stars over your head are a mosaic in time – the light is all hitting your retina at the same time, but (in the summer sky) you’re seeing how Altair looked in 2001, how Vega looked in 1983, how Antares looked in the year 1398, and how Deneb looked in about 600 BC. Let’s not even get into the deep-sky objects – if you stay up a bit later and can see the naked-eye fuzzball of the Andromeda galaxy, that light is from around the time that the australopithecines were learning how to spend more of their time walking on two legs.

Similarly, when you look at a DNA sequence, you’re looking at a mosaic of conservation. Some parts of that string of letters are nearly unchanged all the way from humans to sea cucumbers, while others are different between every human on the planet (OK, short of identical-twin human clones, I suppose). So to be technical about it, there is no such thing as “the human genome”: everyone has their own list of variant sequences, most of which make little or no difference because they’re in genomic regions that can handle them. That’s why we talk about “consensus sequence”.

Cancer cells are at the far end of that scale, though. The more unstable ones are throwing off mutations constantly, by all sorts of mechanisms – and, of course, some of those are incompatible with life, but the ones that survive just keep on cranking. What you see when you look at a tumor sample are the lineages that have made it, that have (unfortunately) managed to randomly stumble into being the best tumor cells that they could manage to be, and the disconnect between their priorities and the priorities of the rest of the body are the disease that we call “cancer”.

Here’s what long-read sequencing revealed in this case (ERBB2 is also known as HER2):

Now we have applied long-read sequencing to explore the hidden variation in a cancer genome and have discovered nearly 20,000 structural variations present, most of which cannot be found using short read sequencing and many are intersecting known cancer genes. More than twice as many of the copy number amplifications could be explained through long-range variants identified by long-read sequencing compared to short-read sequencing. We further found the ERBB2 oncogene to be amplified through a complex series of events initiated by a large translocation into the highly rearranged hotspots of Chr 8, where the sequence was then copied dozens of times more with further translocations and inverted duplications resolved only by the long reads. Furthermore, we find 20 additional inverted duplications throughout the genome, highlighting the importance of this underreported structural variation type. Overall, using long-read sequencing we see that far more bases in the genome are affected by structural variation compared to SNPs.

There are several ways for those inverted duplications to happen (just to pick one of the problems), but in general, given the hosed-up nature of DNA handling, quality control,  and cellular death checkpoints in a cancer cell, pretty much anything that you can imagine happening is probably happening at some point. It’s especially interesting to see the complexity of what’s happened to HER2 – if there’s a popular perception at all of DNA problems with cancer, it’s likely a picture of one little thing going wrong. But the reality is that stuff goes into a combination duplicator/industrial fan, and gets flung all over the genome. Note that in these cells HER2 has been translocated into chromosome 8 from its original home on chromosome 17 and then copied “dozens of times” with more translocations. We’re seeing the result of a long series of bad events piled up on each other. To paraphrase Adam Smith, there is a lot of ruin in a cell.

21 comments on “A Close Look at a Cancer Genome”

  1. Isidore says:

    “Craig Venter ≠ humanity at large”
    Craig Venter would be to differ, I suspect.

    BTW, the CAPTCHA math problems have become more difficult as of late, involving multiplication of larger single digit numbers, it used to be addition and subtraction primarily.

    1. Wavefunction says:

      The CAPTCHA option is controlled by an increasingly malevolent and clever AI. In a very short time, only those familiar with advanced math concepts like Bessel functions and the Riemann- Roch theorem will be able to comment on Derek’s blog.

      1. matt says:

        Oddly enough, soon only our AI overlords will be able to get past the bot filter. Just as in most cases only our computer overlords can remember all the passwords and security strings and PINs needed to secure our computing information. I plan to brush up on my Bessel function solving so I can be the last squishy standing, in order to gain supervisory standing in the sugar mines where we are enslaved. (Nods to Homer Simpson.)

      2. Nick K says:

        Why is the CAPTCHA fuse so absurdly short? I find I have to renew it at least once, even for a short post.

        1. zero says:

          You can click the refresh button next to it and get a new problem without needing to retype your post.

  2. cynical1 says:

    I always thought the Andromeda galaxy was really far away. I’m not sure that’s a good analogy. It’s not like australopithecines are extinct. They have an orange head and work at the White House.

    1. secret sauce says:

      No need to denigrate australopithecines or orangutans by what homo sapiens hath wrought.

      I like the analogies between genomics and astronomy. The final frontier?

      1. Chairman Mao says:

        That orange orangutan was responsible for the largest increase in NIH funding in a decade, so keep up the derogatory ape comparisons-It didn’t do Roseanne Barre any good.

        But question 1: German gene masters say The human genome is 25% flawed? Good place for a crowd sourced opinion.

        Q2: Increased CAPTCHA complexity an attempt by Science to screen out the Hoi polloi?

        1. cynical1 says:

          You should get your facts straight Chairman Mao. On May 23, 2017 the Putin Poodle presented his budget proposal to Congress which called for a $6 billion cut (22%) to the NIH. Look it up. You can thank the Congress that he did not get it. Not him. Face palm.

        2. tim Rowledge says:

          I’m afraid that you’d be filtered out for not knowing that hoi polloi needs no definite article. Apparently.

          1. Derek Lowe says:

            That’s one of those pieces of knowledge that I came across some years ago and have since wondered if I’d be better off without – since now I just notice it all the time (!)

    2. Nick K says:

      The Orange Australopithecine also made a huge fortune in the Manhattan real estate market, something the vast majority of H. sapiens could not do.

      1. johnnyboy says:

        Any australopithecene could have put the orangehead’s daddy’s present of 20 million into bonds and made more money than what he has now (without having to declare bankruptcy four times in the process).

  3. Imaging guy says:

    Whenever you read a CRISPR article, they report that the sequencing shows that CRISPR is specific. Recently, a group shows by long read sequencing that CRISPR can lead to mutagenesis. “Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases.” (1) This article was widely covered in news and led to the drop in stock prices of CRISPR companies. (2)
    1) “Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements”, 2018, Nature Biotechnology

  4. luysii says:

    SK-BR-3 HER2+ has been in use for a while (not sure how long). Cancer genomes are known to be quite unstable. It would be of interest to perform the same long reads on a frozen SK-BR-3 HER2+ cell line of a few years ago. How much of what we’re seeing is the result of continued proliferation in culture?

  5. Charlie Kilian says:

    The light reaching us from far away galaxies like Andromeda (itself fairly close as those things go!) is even crazier than you make it out to be. We think of Andromeda as one thing, as if the light reaching us from Andromeda all left it at the same time. But the Andromeda Galaxy itself is about 220,000 light years across, and it is facing us tilted at an angle. The light from the far side of the galaxy is taking significantly longer to reach us than the light from the near side. And that’s true of most galaxies and galaxy clusters — all of them that aren’t oriented at just the right angle. And yet, for most purposes, astronomers ignore that vast (-in-everyday-human-terms) distance because it’s negligible to what they study.

  6. Mike says:

    As a chemist, it’s been a long time since I took a biochemistry class, but let me check my thinking.

    If cancer cells see such a high number of mutations, I assume that cancer cells are throwing out a plethora of bizarre proteins when the genes are expressed? Normal genes that might produce insulin are mutated to give a ton of different insulin fragments (if the mutation doesn’t prevent expression). And unless the cell marks these useless proteins for destruction, they can end up circulating around the body?

    Is there any data that’s look at protein express in cancer beyond just the key biomarkers that are common across tumor variants?

    1. Nesprin says:

      Yes, there’s tons of work on the altered proteins in cancer. Bcr-Abl in chronic myeloid leukemia is a great success story from this approach- a single protein, unique to cancer found in all CML patients which when blocked cures the cancer. Problem is, solid tumors are not hematologic cancers- instead of a small number of stem cells giving rise to the bulk, you’re facing a mess of different related cell types of different genome, potency and niche. Thus proteomics in solid tumors frequently stalls for horrific signal to noise problems- there’s so many changes in so many different parts of the tumor that figuring out what matters is beyond hope most of the time.

  7. JB says:

    On an interesting side note, go look up the work by Chad Slawson out in Kansas. There are very compelling and direct links between metabolic reprogramming like the long observed Warburg Effect and chromosomal instability.

    J Biol Chem. 2013 Sep 20; 288(38): 27085–27099.

    J Biol Chem. 2010 Nov 5; 285(45): 34460–34468.

  8. JG4 says:

    @Charlie Killian – It is even more disturbing than you suggest. In the reference from of the photons originating in the Andromeda galaxy and all of the others, emission (or creation) are simultaneous with absorption (or annhilation). The photons that mediate quantum electrodynamics do not experience time. Apparently, matter, but not energy, experiences the flow of time.

    Richard Feynman’s Lecture On Entropy (Part 1)

  9. JG4 says:

    that should be “reference frame”

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