Liam’s Passing

This entry is part 10 of 10 in the series "Liam's Battle" --

2Harris-8vin1Sept2015 – Liam took his last breath at 4:36 am. His departure from this life was peaceful. Fred and I were by his side.

2 Timothy 4:7-8 “I have fought the good fight, I have finished the race, I have kept the faith. Now there is in store for me the crown of righteousness, which the Lord, the righteous Judge, will award to me on that day-and not only me, but also to all who have longed for his appearing.”

Topotecan

This entry is part 1 of 1 in the series "NB Chemotherapy" --

WARNING: Science Content


As with all my posts, I try to include some thought provoking science, as well as interesting water cooler talk.  Good luck.
 

Topotecan

Prerequisite Reading

Some DNA Basics

DNA Replication and Torsional Strain


Introduction

Liam’s Rounds 1 and 2

The first chemotherapy drugs in Liam’s treatment were topotecan and cyclophosphamide. He received two five-day treatments with these drugs.  The first was during Aug 2014 and the second was in Sept. 2014 (separated by four weeks).

The drugs are given together for a combined treatment that offers a synergistic effect.  In his case, they proved to be very effective.  His cancer could not be found in a PET scan after just one round. (This doesn’t mean that his cancer wasn’t present, just that the much of the solid mass tumors were no longer taking up glucose … in other words – dead or dying.)

 Focus

Even though chemotherapy drugs are often given in pairs to give a compounded blow to the cancer, in these blog posts I will explain the mechanism of each drug individually so that it can be digested by the audience.

Fig 1. Topotecan

Topotecan

Topotecan is a topoisomerase I inhibitor which is derived from a plant called the Asian “Happy Tree” (Camptotheca acuminata).[1]  Topoisomerase I, discussed in a previous blog post,  is an enzyme that relieves torsional strain ahead of the advancing DNA replication fork. Without this torsional strain relief, the DNA is too strained to be able to separate into two strands.

A chemical mechanism for how topotecan inhibits topoisomerase I is proposed in the Proceedings of the National Academy of Sciences. [2]  The authors suggest that as the one side of the DNA is pulled apart by topoisomerase I, the topotecan molecule wedges (intercalates) between the +1 and −1 bases of the duplex DNA, and is further stabilized by six different protein contacts while the DNA is open. (Think of jamming a zipper, and then sewing the thing that is lodged in the zipper in place.)

Ribbon Diagram of regular Topoisomerase I breaking one side of the DNA

Fig 2 – Ribbon Diagram of regular Topoisomerase I breaking one side of the DNA, used by permission [3] PNAS, 99, 24, pp. 15387- 15392

The intercalation of topotecan causes a shift of the downstream bases by ~3.6 Angstroms (defining the displacement that this monkey wrench causes in the system). Figures 2 and 3 show ribbon diagrams of both the regular process and the process that is poisoned by topotecan.

Ribbon Diagram of Topoisomerase I with Topotecan interfering with the process.

Fig 3 – Ribbon diagram of topoisomerase I with topotecan interfering with the process, used by permission [4] PNAS, 99, 24, pp. 15387- 15392

An article in Nature [5] provides evidence that topisomerase I is inhibited (poisoned) more readily when the forming supercoil has a positive coiling direction. Topotecan has the effect of being a monkey wrench in the topoisomerase I process.

In slightly easier to understand terms, the topotecan is just the perfect shape with just the right bonding attachments to act like something getting stuck in a zipper.  Eventually, with enough vibrations and thrashing about, the topotecan will get unstuck, but cancer is impatient with the process and typically triggers the apoptosis alarm (cell death trigger) before it can work the topotecan out of the zipper.

License Number  3681730918315, 8/4/2015, NATURE PUBLISHING GROUP LICENSE

Fig 4 – Cover art that shows a good overall picture of the topotecan poisoning process. Reprinted and adapted by permission from Macmillan Publishers Ltd: Nature, 448, 213-217, copyright 2007

 

 

  1. http://chemocare.com/chemotherapy/drug-info/Topotecan.aspx 
  2. PNAS, 99, 24, pp. 15387- 15392 (26 Nov. 2002), “The mechanism of topoisomerase I poisoning by a camptothecin analog”PNAS Cover
  3.  Anyone may, without requesting permission, use original figures or tables published in PNAS for noncommercial and educational use (i.e., in a review article, in a book that is not for sale) provided that the original source and the applicable copyright notice are cited. http://www.pnas.org/site/aboutpnas/rightperm.xhtml
  4. See note 3
  5.  Nature 448, pp. 213-217 (12 July 2007), “Antitumour drugs impede DNA uncoiling by topoisomerase INature Artwork

DNA Replication and Torsional Strain

This entry is part 2 of 2 in the series "DNA" --

WARNING: Science Content


As with all my posts, I try to include some thought provoking science, as well as interesting water cooler talk.  Good luck.
 

A Schematic / Block Diagram

DNA_replication_en.svg

Many enzymes work together in the replication of DNA. The focus of this post will be on the topoisomerase enzymes and their function. In another upcoming post, I will introduce topoisomerase inhibitors. It is important to understand what is going on with these enzymes and how the topoisomerase enzymes work to relieve torsional strain during DNA replication.  

Torsional Strain

When DNA is at equilibrium and not under any torsional strain, one full rotation of the double helix contains 10.6 base pairs.  When it is replicated, the entire DNA strand has to be divided into two new strands. This separation introduces a significant amount of torsional strain in the DNA coil. (Think of that handset cord on your desk phone that is completely tangled up because of all of the rotations of the handset over time.) This process of DNA entanglement is called supercoiling.  The only way to relieve the strain is to break the DNA, pass a segment through, and put it back together.  The topoisomerase enzymes do just that. They relieve the mounting torsional strain before the strain becomes so great that the replication process is halted. Still confused? Take a look at this video.

Some DNA Basics

This entry is part 1 of 2 in the series "DNA" --

WARNING: Science Content


As with all my posts, I try to include some thought provoking science, as well as interesting water cooler talk.  Good luck.
 

Deoxyribonucleic acid (DNA)

12 Base Pairs of DNA. For those of you not familiar with chemistry, but think you know what an atom is, each stick in this figure represents a bond between atoms. The color of the stick represents the atom that is being bonded to. If the stick line changes from one color to a different color, then you are bonding two different types of atoms together. (i.e. Grey to White = Carbon bonded to Hydrogen) You’ll also notice that some atoms bond to multiple things and others not so many. How many things it attaches to is determined by where it is on the periodic table of the elements. (Don’t worry about this, just appreciate that different atoms bond to different numbers of other atoms.)

There Is a Lot of It.

As we have mentioned before in a different post, there are about 3 billion base pairs of DNA over 46 chromosomes (23 pairs).  This count is for each cell in your body. You have about 70 trillion cells in your body. Yes, that is 70,000,000,000,000 copies of your complete DNA. (For those of you heady in math, that is 210,000,000,000,000,000,000,000 base pairs in your body right now.)

DNA Is a Storage System

DNA is an elegant storage system, and every time that I stop and think about what it is, I marvel at its simple, yet complex nature. The rotating picture to the right is 12 base pairs. The two separate helices are the attachment points of each side of the base, and the base pairs are the flat planes of atoms that connect one helix to the other. (Go ahead and take a moment and convince yourself that there are only 12 base pairs here. I know that you want to!)

The base pair that is in the center is not “technically” bonded. They are held together with a much weaker force than the standard covalent bond.  They are held together with “hydrogen bonding“.  Hydrogen bonding is an electrostatic force that only exists when hydrogen is bonded to a Flourine, Oxygen, or Nitrogen atom.  When hydrogen is taking part in hydrogen bonding, it acts like a strong magnet would if it were fastened to one piece of metal, and another piece of metal got close to it. DNA would be useless without this weak interaction.  It needs to be able to separate easily.  If it were covalently bonded across the base pairs, it would be useless.  (If you look closely at the rotating DNA picture, you will notice that the base pairs are shown with a break between them. The hydrogen bond between them is just strong enough for the base pairs to line up to one another.)

 

A closer look at base pairs

A Closer Look at the base pairs (Thanks to Zephyris on Wikipedia). Each of the four base pairs have names, but to keep it simple, they are abbreviated with the letters A,T,C, and G.  —“A” hydrogen bonds to “T”  — “C” hydrogen bonds to “G” — Also notice that the hydrogen bond between the base pairs is represented by a dashed line. (i.e. Not quite as strong as a regular old covalent bond)

 

DNA Must Break Apart

As a storage system, DNA must continually break apart in order to be read or to be duplicated.  When it is in its double helix form, it is pretty. It is in storage. It just sits there. Hydrogen bonding (which is not nearly as strong as a regular bond) can break apart easily. It can be put together easily. There are two main events that occur with DNA. Copying the DNA strand, and reading the DNA in order to produce the biological machines in the human body that are comprised of proteins.

The following videos describes how the DNA is broken apart and either copied or transcribed.

The Focus of Many Chemotherapy Agents

The main Chemotherapy drugs that are used (at least for Neuroblastoma) focus on the interruption of the copying process. An interruption of the copying process triggers the oncogenes in the cell to cause apoptosis (cell death). While healthy cells have the means and time to do their best job at repairing the cell before sounding the apoptosis alarm, cancer cells are not nearly as patient. When they discover that there is a problem with DNA replication, they will often give up… At least until they assimilate the knowledge of how to repair the DNA or the knowledge of how to keep the chemotherapy drugs out of their cell walls. This adaptation leads to the demise of the effectiveness of chemotherapy drugs in their fight against cancer.

mIBG – We are back on!

This entry is part 3 of 3 in the series "Radiation" --

WARNING: Science Content


As with all my posts, I try to include some thought provoking science, as well as interesting water cooler talk.  Good luck.
 
MetaIodoBenzylGuanidine

MetaIodoBenzylGuanidine


[Prologue]

For those avid followers of all of the science that I have introduced during all of this, you will recognize that this post is a rehash, but an exciting rehash.

Early on, Liam was initially slated to participate in a mIBG (MetaIodoBenzylGuanidine) trial.  The trial wasn’t supposed to happen until after the induction period of chemotherapy.  The catch is that only 80-85% of neuroblastomas absorb mIBG.  This is why fairly soon after diagnoses, while there is still a lot of cancer in concentrated pockets throughout the body, an mIBG scan is given to see if the particular variety of neuroblastoma picks it up. This is done with [123]- mIBG (see below.)

When this scan was performed on Liam back in September, there had been problems and the scan was not performed right away.  The doctors and hospital staff were having difficulty stabilizing Liam.  In fact, that first session in the hospital lasted over 21 days.  He had been started on  a fantastic chemotherapy drug called Topotecan (which is a TopoIsomerase I inhibitor that is so darn cool, it deserves its own post and I will not discuss it here.)  The problem was that Liam was spiraling out of control while he was on it. He started having trouble breathing, and he ended up with a plural effusion (yep… that was a bad couple of days. Click here for the post from that day).  After all was said and done, the mIBG scan was pushed off until he was admitted for chemo round 2.

When the [123] mIBG scan was finally performed, it came back negative. We were bummed, but the chemo seemed to be going so well that we really didn’t give it much thought. Liam was feeling better.

After 6 rounds of chemotherapy, a follow up PET scan was performed. No cancer showed up on the scan. He had a remarkable response, and we thought we were doing pretty well.  In reviewing all that had happened over the course of 6 rounds of chemo, I wondered if most of the cancer had vanished just after the first round of chemotherapy.  If it did, it would have skewed the mIBG test to a negative result.

Looking back at the sudden improvement after round 1- the plural effusion (now believed to be caused by cancer dying his lungs) and all of the immediate weight loss (now believed to be the cancer dying in his abdomen)…. he looked normal for the first time in months; I contend that the Topotecan chemotherapy made most of the cancer disappear quickly.  His response even astonished his doctors.

Now that the cancer has come back,  it was suggested by the doctors at CookChildren’s that we look one more time at the mIBG.  So, we did, and it gave a positive response to mIBG.  It can clearly be seen in the left tibia and the pelvis.

mIBG Liam 24July2015

123-mIBG Scan of Liam on 24July2015. His neuroblastoma has soaked up the mIBG compound, and due to its radioactivity is exposing the film. His trouble spots in his left leg and pelvis can clearly be seen.

So what does this mean? It means that we now have a really awesome tool in our tool chest to fight this. It won’t cure the neuroblastoma, but hopefully we can knock it down and coupled with other therapies, we can get this disease under control for Liam.  This is an option that a week ago we did not have.

[End of Prologue]

So, how does it work? It turns out that Neuroblastoma has a strong affinity for mIBG in about 85% of cases.[1] In a very high percentage, the Neuroblastoma cells will take this compound up while the normal cells will not. This is called ‘selectivity’. (i.e. the Neuroblastoma soaks this compound up selectively over normal cells).  mIBG in itself, however, doesn’t do anything. It is taken into the cell, and then is excreted from the cell at a later time.  This means that the Neuroblastoma cells are not sensitive to the compound. 

A clever and ingenious pupil of chemistry can already see what to do next.  Swapping out the Iodine atom on this compound with the radioactive version  makes this molecule very useful.


[123]-Iodine

123 Iodine will decay by electron capture to form 123 Tellurium which will then emit a Gamma ray with an energy of 159 keV. This is useful for imaging.  This is like having an x-ray performed, but rather than having an x-ray source shining high energy light through Liam, the light will be generated inside him!  Since this radioactive atom is attached to a compound which is only selective to Neuroblastoma, Gamma rays (like x-rays) will be generated only at the Neuroblastoma sites.  With the correct detector, the Neuroblastoma will light up like a Christmas tree.


[131]-Iodine

If 131 Iodine is used, different results will be observed.  131 Iodine decays in the follow two manners (statistically a 90% Beta(-) Decay and a 10% Gamma decay):

(Beta(-) Decay ~90%) {^{131}_{53}\mathrm{I}} \rightarrow \beta + \bar{\nu_e} + {^{131}_{54}\mathrm{Xe}^*}  + 606 keV 

(Gamma Decay ~10%) {^{131}_{54}\mathrm{Xe}^*}  \rightarrow {^{131}_{54}\mathrm{Xe}} + \gamma  + 364 keV

The Beta(-) decay produces a very energetic electron and an Antineutrino which have a tissue penetration of about 0.6 to 2 mm. This is enough energy to destroy cells. (i.e. a cell sized atomic bomb) So in essence, this gives a pathway for the mIBG, which is very selective to the Neuroblastoma, to blow up the cells (and leave the good cells alone).


This mIBG scan that was performed today only involved 123 Iodine for gamma ray imaging (see above images). This indicates all of the places that the Neuroblastoma is, with a few exceptions. There are false readings in some of the places like the thyroid (which regulates Iodine containing compounds).    In the coming weeks, it’ll be time to bring out the [131] Iodine and give this cancer the radioactive punch it deserves.

  1. According to NationWideChildrens.org “…Roughly 80-85% of neuroblastomas will absorb MIBG. There are really 2 ways in which MIBG treatment is used. In both methods, the MIBG chemical is attached to an iodine molecule that has been made radioactive. The radioactivity can be either a low-dose or a high-dose…. ”

Gene Blues and MYCN Amplification

This entry is part 9 of 10 in the series "Liam's Battle" --

WARNING: Science Content


As with all my posts, I try to include some thought provoking science, as well as interesting water cooler talk.  Good luck.
 

Neuroblastoma Genetic Testing

There is a lot of stuff that is not known about Neuroblastoma, but there are a few key markers on the genetic sequence that have had some statistics gathered and can provide an inkling of whether or not the particular breed (or genetic makeup) of the Neuroblastoma is going to be more (or less) difficult to fight. The list I have here is not comprehensive. I am not a medical doctor. So, I offer my own interpretation, which I may find later to be inaccurate. If I do find inaccuracies, I will correct them. I am a chemist, but biochemistry is not my specialty. I am a material scientist, so there is a good chance I will get some of this wrong. I have approached this with the background that I have and with the attitude that I am a dad who needs to understand what the heck is going on, and I am learning as fast as I can!

I have included a *brief* reminder on the genetics of the human body for those audience members who have (ahem) forgotten some of their high school biology.  Also, there is a lot of blockquotes in this post. Feel free to follow the references and see just how deep the rabbit hole goes. 🙂

What are genes?

BY-SA 3.0 Zephyris

Genes are like the lines on a blueprint for living things.  Your genes are in-fact what make you distinctly a human and not a cat, dog, or dinosaur. All living things have genes. Genes are the sequences of DNA that instruct the cell on how to build protein or peptide sequences. Genes (DNA sequences) are all lined-up into libraries called chromosomes. The human body has 23 chromosome pairs (46 chromosomes, half from mom and half from dad).  No matter which cell you look at within one individual, whether it be from the big toe or the liver, each cell in a body has a copy of all of the chromosomes.  There are approximately 3 billion base pairs (Yes…~3,000,000,000) required to make the 46 chromosomes in each cell. That is a lot of copying of base pairs that is going on all the time. It is quite phenomenal that it happens correctly once, much less regularly in the ~37 trillion cells in an adult human.

Neuroblastoma Genes – As far as we know, what can go wrong?

Well, I think that it is pretty clear that there is a lot that is not known about what goes wrong in cells that have turned into Neuroblastoma. There is currently no known link to any cause, and to make matters even more confusing, some kids can actually get Neuroblastoma cells, and at a later date be completely free of them without any treatment. (This only happens with Stage 1 low risk Neuroblastoma. There are no known cases of Stage 4 high risk Neuroblastoma suddenly disappearing.)

Given all of this, however, there are some things that can be determined from a genetic test of the Neuroblastoma cells. The primary reason for testing the cells is to estimate how challenging the fight will be for a patient’s particular Neuroblastoma.

 MYCN Amplification

The  MYCN protein regulates fundamental cellular processes from proliferation to apoptosis (cell death). If the cell has more than 10 copies of this protein, it is bad. So bad, in fact, if Neuroblastoma cells have this, then it is automatically classified as high-risk (even if it were discovered early and would otherwise be classified as low-risk). The MYCN protein genetic information is located on chromosome 2p24.3 between base pairs 15,940,560 and 15,947,006. [Ref]

MYCN is a protein and a member of the MYC family of proto-oncogenes.  A proto-oncogene is a normal gene that has the potential to become cancer because of mutations or some type of increased expression (expression is how information from a gene is utilized to create another genetic product such as a protein).  “Like many other MYC proteins, MYCN is a transcription factor that controls expression of many target genes, which in turn regulate fundamental cellular processes including proliferation, cell growth, protein synthesis, metabolism, apoptosis and differentiation” [Ref]

Having too many copies of the MYCN protein makes this type of cancer hard to fight, but some interesting findings actually show that the MYCN protein may in fact be involved in the creation of the Neuroblastoma [Ref], although this is disputed other places [Ref]. It is possible that there is a system of checks and balances with some of the genetic information on chromosomes 1 and 11, which may help regulate over duplication of the MYCN protein, and the amplification of MYCN is closely related to  “missing” information on these Chromosomes. (Also called a ‘deletion’)

Deletion From Chromosome 1 and Chromosome 11

The deletion of a chromosome implies that there are areas of the genetic code, which we know are typically present in the DNA, that are missing from the sequence of the person whose DNA is being examined. Deletion from chromosomes 1 and 11 seem to be linked to to Neuroblastoma:

… Researchers believe the deleted regions in these chromosomes could contain a gene that keeps cells from growing and dividing too quickly or in an uncontrolled way, called a tumor suppressor gene. When a tumor suppressor gene is deleted, cancer can occur. The KIF1B gene is a tumor suppressor gene located in the deleted region of chromosome 1, and mutations in this gene have been identified in some people with familial neuroblastoma, indicating it is involved in neuroblastoma development or progression. There are several other possible tumor suppressor genes in the deleted region of chromosome 1. No tumor suppressor genes have been identified in the deleted region of chromosome 11. [Ref]

About 25 percent of people with neuroblastoma have a deletion of 1p36.1-1p36.3, which is associated with a more severe form of neuroblastoma. Researchers believe the deleted region could contain a gene that keeps cells from growing and dividing too quickly or in an uncontrolled way, called a tumor suppressor gene. When tumor suppressor genes are deleted, cancer can occur. Researchers have identified several possible tumor suppressor genes in the deleted region of chromosome 1, and more research is needed to understand what role these genes play in neuroblastoma development. [Ref]

The Gene List: (Changes in These Genes are Associated with Neuroblastoma)

 

Liam’s Results

Fluorescence in-situ hybridization (FISH)

When Liam was in the hospital the first time, a sample of the neuroblastoma was collected from his bone marrow before he began chemotherapy. This sample was sent off and analyzed by the FISH method. (Specifically the report says “FISH for MYCN (2p23-24) gene amplification,” which we know from above is one of the important mutations that predicts poor prognosis.)

Out of the 200 cells probed for MYCN Amplification by the FISH technique, 16 showed to be abnormal, and 184 showed to be normal. Indeed, his Neuroblastoma cells show MCYN amplification.

Chromosome Analysis by Karyotyping

Karyotyping is a way of analyzing the chromosomes for number and completeness. The results from Liam’s test are as follows:

  • 21 cells were counted and analyzed, and 3 of the cells were karyotyped.
  • 19/21 cells were of normal male chromosome compliment
  • 2/21 cells showed a gain on Chromosome 7 and multiple “double minutes

The gain on Chromosome 7 and extra fragments of DNA material are likely a direct cause of the amplification of MYCN gene.

Further Discussion

Now, this is discouraging news, and the words “poor prognosis” have bounced around my cortex for a while. What do we make of this? How can we put this all in perspective?

I was encouraged by a Japanese article which investigated the use of blood stem-cell transplantation (SCT) to treat Neuroblastoma (SCT is a procedure that Liam is scheduled for early next year). It would appear that SCT increases the odds of survival of Neuroblastoma patients with MYCN Amplification significantly (from the low 20% to about 50% survival after 66 months). The article concludes with the following statement:

Not all patients with advanced neuroblastoma who have more than 10 copies of MYCN will die. The requisites for survival in such patients seem to be intensive induction chemotherapy, effective surgery, irradiation, and the use of SCT.  [Ref]

Positive things to consider:

  • This test did not show Chromosome 1 or 11 deletion.
  • The DNA gain was in Chromosome 7 and in double minutes. Chromosome 7 is not tied to anything normally seen with Neuroblastoma. So it is possible that the MYCN has amplified a part of the DNA that might be easier to fight than a Chromosome 1 or 11 deletion (which takes away some of the regulation of MYCN). We will have to wait and see.

Between Rounds 2 and 3

The PET Scan, a Chemist’s View

This entry is part 2 of 3 in the series "Radiation" --

Liam had a Positron Emission Tomography (PET) scan on 19Sept2014. For this the radioactive tracer is Fludeoxyglucose (18F) or (18F-FDG) for short. For those of you crazy chemistry people out there (like Jenn and myself), check this out:

a)Fludeoxyglucose b) β+ Decay of a proton emits a positron (and changes Fluorine to Oxygen); Since a positron is the antimatter equivalent of an electron, when it finds the closest electron it will annihilate. The matter will cease to exist, and it will turn into energy in the form of light (gamma rays). The two gamma rays produced each will have 511 keV of energy. c) With a little acid, the product will be glucose and continue through the energy cycle in the cell. Until the radioactive decay, the molecule is stuck. There is no chemistry available to the cell to process the glucose with substituted Fluorine, once the F gets converted to a hydroxyl, the chemistry can proceed as normal (With a heavy, but stable Oxygen atom)

The 18F-FDG looks just like the glucose molecule except for a heavy fluorine in place of the  2′ hydroxyl group.  Since all cells use glucose as a power source, the PET scan exploits the fact that cancer cells require more energy, and they will take up more of the compound than normal cells. The areas of the body emitting large amounts of gamma radiation are likely to have concentrations of cancer cells.

For those of you (and I know who you are) who would like even more information, here are some links that I found helpful when I was coming up to speed on the technique:

Fludeoxyglucose (18F)

Positron Emmision Tomography

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications (scroll down to figure 13)

Decay scheme of 18F