Thoughts so far . . .

Let’s recalibrate:

At the start of this blog, I had a clear goal: to share breaking cancer research in a manner that is easy to understand.  Who knew – it’s harder than it might seem!  The intricacies of cancer combined with the complicated technical jargon and methods make this task somewhat difficult, particularly if the goal is to break down the science into meaningful pieces of information without taking away from the science.

And so, I find that the beginnings of this blog has become more educational and perhaps more useful for entry-level students hoping to break into the cancer research field.  This information is still valuable for the general public and those merely curious about cancer and cancer research, but for them, it might take some slugging to get through the articles.

And so, I’m dividing this blog into two parts: one is basic informational posts about techniques or topics relevant to cancer, without focusing on newly published research; the other part will highlight new cancer research in a more newsy manner.  The former will be similar to what’s already been discussed, with more explanations and introductions to key players in the cancer field.  The latter section aims to highlight major findings without going into all the details.  My perspective on these topics can still be found in the “Where do we go from here” section.  You'll know by the title (Educational vs Research) where today's topic falls.

The combination of these two parts will hopefully give you, my readers, the information necessary to critical evaluate the science for yourself and to see the progress being made in the cancer research field.

This new direction is a challenge: a challenge for me to write in a clear, concise, and truthful manner; and a challenge to my readers to keep reading.  More than that, ask questions.  What do you want to know about?  Is a term or concept not understood? Do you disagree with my perspective?  With that, we can be assured we are in this endeavor together.

The Beauty of Cancer

Most people would not describe cancer as beautiful or elegant, particularly those of us that have survived the disease.  Even more, battling cancer may affect your sense of beauty, may alter your own body image. 

Jacqueline Firkin, associate professor in theater and film at the University of British Columbia wanted to give women something beautiful, something that opened the discussion surrounding cancer, beauty, and body image, something women could identify with.  She created 10 ball gowns fashioned after microscopic images of cancer cells and cellular processes hijacked in cancer.  The results are beautiful.

Next Generation Sequencing in the Laboratory

With next generation sequencing (NGS) comes the power to change medicine, to personalize patient treatment, to improve patient outcomes.  But before we become overly optimistic, recall what we learned in our last discussion – that use of NGS in the clinic possesses some limiting factors, one of which is our incomplete understanding of cancer.  For personalized medicine to be effective, cancer scientists need to have an in-depth understanding of the biology behind cancer.  For those of us toiling away in the research field, utilizing NGS has become a new tool for deciphering intricate cancer networks.

In today’s discussion, we’ll uncover how a group of researchers based in Portland Oregon used NGS to assess the RNA levels or transcript levels of genes controlled by the tumor-promoting oncogene c-MYC in a breast cancer cell line (for an explanation of the relationship between RNA and DNA, look here).  In this report, the use of RNAseq (the NGS technology used to looked at RNA levels) composes only a small, though critical, fraction of the overall study.  It also represents the utility of NGS technology in basic research.

Personalized Medicine: Next Generation Sequencing in the Clinic

Now that we’ve established the basics behind next generation sequencing (NGS), we can more fully delve into its implications.  If you missed the last post, check it out here.  In the current post we’ll uncover if and how NGS can be used in the clinic.

The need to sequence DNA faster and cheaper stems partly from our desire to impact patient diseases.  The future of cancer therapy lies in our ability to target pathways (and the proteins involved) that are altered in cancer cells.  But as we’ve shown, each cancer is unique and the genes involved in that process can vary between patients and even within that same patient.  In a recent publication, using next generation sequencing techniques, the authors analyzed a series of patients with pancreatic cancer and assessed whether looking at them as a group versus looking at their mutations as individuals was more advantageous when deciding treatment options.  They compared pathways altered in individuals to those that were significantly altered across the group and found little overlap suggesting that grouping patients may not provide the most valuable information when deciding treatment options¹. Therefore, personalized medicine, or individual gene expression profiling is critical to the acquisition of clinically significant information.  And the ability to do this lies in next-generation sequencing.

Reading Genomes: A brief history of next-generation sequencing.

We’ve talked a lot about DNA, genes, and the identification of specific mutations in specific genes related to cancer progression.  But how is this done?  What is the methodology?  Why should you care? What are the practical applications for research? For patients? What are the limitations? 

The technology we are talking about is called “next-generation sequencing” and it is used to decipher the genomes of entire organisms or, for our purposes, the genome of cancers.  In fact, the genetic differences between cancer types or those responsible for intratumor heterogeneity, as we previously discussed (see Mutational Landscape of Cancer, Intratumor Heterogeneity), were identified through the use of next-generation sequencing.  In this three-part discussion, we will first learn about the methodology involved in this latest technology and in the following discussion we will delve into its implications for both researchers and patients.

Imagine your genome or your cancer genome are like books, composed of a series of letters, our DNA.  There are four letters called nucleotides in our genomic alphabet (A, T, C and G) and like letters, the number of nucleotides and the sequence of these letters create specific words or genes.  Like words organized into sentences and paragraphs, the series of genes are organized onto chromosomes.  Our entire genomic book is composed of 23 chromosomes.  Sequencing the entire genome of individuals or that of specific cancers is akin to reading that book.  And the longer the book, the more difficult it can be.  Additionally, spelling mistakes, or mutations can further complicate the reading.

Our ability to read through genomes, to sequence the genes and the DNA that makes up those genes is based on our understanding of the structure of DNA.  Thanks to Watson and Crick, we know that DNA is configured as an anti-parallel double helix.  Essentially this means DNA consists of two strands that twist together in a head-to-toe fashion.  Think of it like a zipper, each half binds, connects, to the other half to make a tightly closed structure.  The most critical aspect of this structure lies in the fact that the two DNA strands are complementary.  Through experimentation, we know that each of the 4 nucleotides binds each other with precise specificity: A only binds T and C only binds G.  Thus, if we know the sequence of one strand, we can easily deduce the sequence of the second.

Sanger Sequencing: the Gold Standard

For the last 35 years, the gold standard for sequencing has been Sanger sequencing also referred to as the chain termination method.  Although next-generation sequencing (NGS) has taken this knowledge to the next level, allowing for analysis of large genomes in a quick and accurate manner, Sanger sequencing is still widely utilized for its simplicity and cost-effectiveness.  To understand how NGS has improved our ability to read DNA sequences, we need to understand the methodology behind Sanger sequencing.

This method utilizes our knowledge of how DNA is replicated in normal cellular processes.  It involves unzipping template DNA (the DNA that needs to be sequenced) to create single stranded DNA and mixing it with a short single stranded complementary DNA strand, called a primer, that specifically binds to the template DNA.  The reaction is started by the addition of an enzyme called a polymerase and a mixture of labeled nucleotides (the letters of our DNA).  The DNA polymerase is the powerhouse behind DNA replication, adding complementary nucleotides (ddNTPs) one by one.  In sequencing reactions, these four nucleotides bases (A,T,C,G) have been altered in two ways: they are labeled (by fluorescence) for identification in downstream reactions, and they are modified so that elongation terminates upon their addition.  By halting the elongation with one of these labeled ddNTPs, the length of the fragment can be utilized for interrogating the base identity of the terminating base.  Future reactions include capillary or gel electrophoresis which essentially separates sequences by length and then identifies the terminating base (See Figure 1). 

Figure 1: Sanger Sequencing¹.

The success story of Sanger sequencing belongs to the Human Genome Project.  Sequencing the human genome required the cooperation of multiple international research institutions and the injection of billions of dollars by governments and private corporations.  After more than a decade, an understanding of what the human genome looks like was generated.²  Such knowledge yields power for understanding the genetic basis behind all diseases from cancer to neurological disorders.  It also helps answer some basic biological questions: why do we taste bitter foods? Why do we see colour? The implications for this technology are huge. However, limitations including speed, scalability, and resolution or accuracy prompted the development of technologies that could sequence larger sequences more quickly and more accurately, and therefore answer more genetic questions.

Next generation sequencing: Illumina

Next generation sequencing is the all-encompassing term for these new methodologies that aim for high-throughput analysis of large sequences such as entire chromosomes or even entire genomes.  Several biotech companies developed NGS methods that differ in their template preparation, method of sequencing and types of analysis.  For an example, let’s highlight the method used by Illumina.

Figure 2: Illumina Next Generation sequencing³.

The first step in this process involves fragmenting the DNA to be sequenced into small fragments. These fragments attach to adaptors which are essentially primers.  This solid surface substrate is propriety for this technology.  The next step involves amplifying these DNA fragments in a manner referred to as bridge amplification.  With the same polymerase used in Sangar sequencing, complementary DNA strands are created.  With this technology, up to 1000 identical fragments can be generated.  This amplifaction uses another propriety element: the incorporation of 4 nucleotides each labeled with a different dye.  Like Sanger sequencing, these modified nucleotides also terminate the reaction after addition to the DNA fragment.  In this way, after laser excitation, the emitted fluorescent signal is captured and the subsequent sequencing reactions can proceed.  These DNA clusters are sequenced, one base at a time and then aligned to a reference sample.

The advantages of NGS compared to Sanger sequencing are clear.  Whereas Sanger sequencing sequences one region at a time, next generation sequencing can sequence multiple fragments simultaneously, speeding up the process tremendously.  Additionally, Sanger sequencing generally only sequences a specific region a limited number of times.  This “read-depth” is dramatically improved with NGS technology whose techniques allow for deep sequencing.  This increases the accuracy of the seqeunce.  With the increased speed and accuracy, the ability to sequence large genomes is now feasible in a short amount of time. 

Once a sequence has been determined it can be mapped to the latest human whole-genome reference using computational algorithms.   The technology to accurately map samples and identify mutations has also dramatically improved in our technological age.  When it comes to using this technology for the identification of mutations in cancers, additional factors need to be considered including germline mutations (what the patient carries independent of the cancer) as well as single nucleotide polymorphisms or small difference in coding DNA.  The interpretation of this data requires intensive knowledge of genetics. 

Stay tuned for the implications of this technology!  We’ll be discussing the role of NGS in the cancer clinic as well as in the cancer research lab.

Today’s uncovered cancer morsel: The advancement of technology directly impacts cancer research and cancer patients.

References

     1.     http://www.eisenlab.org/FunFly/?page_id=24

     2.     International Human Genome Sequencing Consortium.  Initial sequencing and analysis of the human genome.  Nature.   
           2001. 409: 860-922.

     3.     http://res.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf

     4.     Michael Metzker.  Sequencing technologies – the next generation.  Nature Reviews: Genetics.  2010. 11: 31-46

Of Mice and Men: Do men really cause stress?

When you walk in the door, Fluffy (your dog, cat, or pet of choice) jumps up, greets you, voices their approval.  He can sense you; she can smell you; he loves you.  What you may not realize is that when Fluffy senses the men in your house, she might become a little stressed out.

You may have heard about this research article published in Nature Methods recently.  I saw this story on the news: men induce stress in laboratory mice.¹  I laughed, and then I needed to learn more.  So let us take a brief departure from the seriousness that cancer evokes and discuss how and why men cause stress in mice.

The Science:

This study comes out of Montreal and the laboratory of Jeffrey Mogil.  As a pain geneticist, he studies the factors that determine sensitivity to pain.  His lab staff started to notice and anecdotally reported how their presence might affect the behavior of the laboratory mice.  Could this be true? Or just a researcher’s too-critical eye?  The only way to know for sure was to design some experiments to answer the question: do animals respond differently in response to pain when exposed to male and female researchers?

Cancer Commonalities: The Hallmarks of Cancer Part 3

Welcome to the final discussion on the defining characteristics of cancer.  As we’ve discussed in parts 1 and 2 (Part1, Part2) cancer cells share several fundamental traits which are outlined in the figure below. 

Figure 1: Hallmarks of Cancer

In this final discussion, we take a step back:

What allows cancer cells to acquire these traits?  What allows cancer cells to tip the balance on all these processes towards tumorigenesis?  The answer begins with the following two enabling hallmarks.