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Immuno 101


Immunotherapy is a significant game-changer in the world of cancer treatments, and it is beginning to positively impact Cholangiocarcinoma patients. It has been said that 30 – 40% 0f all Cholangiocarcinoma patients have targetable mutations, that could match clinical trials. Please beware of all your options before proceeding with a treatment plan. Immunotherapy may be an option.

“The discovery of the ‘Checkpoint Pathway and its Immunotherapy interventional drugs was a penicillin moment in the history of cancer treatment, we are now seeing the word cure and immunotherapy in the same sentence for the first time.”  Clinical Trials for Cholangiocarcinoma continue to increase.

Helping the immune system recognize and destroy cancer cells

Immunotherapy is a treatment that helps a person’s immune system better recognize and destroy cancer cells. Immunotherapy can encourage your immune system to attack cancer cells. Immunotherapy may have side effects and you should discuss your options with your physician to understand how and if immunotherapy would be a treatment option for you.

If you have a high tumor mutation burden, high levels of microsatellite instability (MSI-H), or mismatch repair deficiency (dMMR), you should consider immunotherapy as a possible treatment. Microsatellite instability and mismatch repair deficiency are types of genetic changes to a cell that interfere with its ability to repair itself. This can lead to cancer cells that are very different from normal cells, which can make them more “visible” to the body’s immune system.

Immune System Overview
Video via CCF Conference 2016

Dr.Kate Kelly – talks about immunotherapy (19mins mark)
She highlights keynote 158 (2015) the first-ever patient to gain a full and complete response (- Rose)

Video collections
What are they?

Checkpoint inhibitors
T-Cells have these on their surface CTLA 4 & PD-1 are the primary examples. Their job is to interact with the cell surface before the attack begins. If the attack is a mistake on a healthy cell they “down-regulate” /switch off the attack – like a fail-safe switch.

Example: PD-1 the inhibitor sits on the T Cells surface, its job is to bind with any PD-L1 present on the surface of a cell (See image below). This is binding communication is friendly indicating a healthy cell and the attack is ceased. PD-L1’s are ligands (elongated proteins) that embed themselves into the cell’s surface and act to stop a T Cell’s act by binding with the PD-1.

Mutated cancer cells have learnt to exploit and hide by expressing PD-L1 on its surface.

Checkpoint Inhibitor Immunotherapy drugs interfere with the T Cells PD-1 inhibitor switch by blocking its function and ability to turn of a T Cell attack.

Check Point Inhibitor Immunotherapy allows our own Immune System to do its job and destroy the previously unseen cancer threat.

It is thought that High Mutation Tumours (TMB -high) express higher levels of PD-L1 ligand proteins and thus exploit this to their advantage.

Currently successful Check Point Inhibitor drugs
Keytruda and Odivo’s  Nivolumab block the PD-1 from binding to PD-L1. Opdivo also has Ipilimumab a drug developed to block CTLA-4 Nivo and Ipi are often used /combined into one treatment.

Other important factors in the success of this therapy are detailed in other chapters “MMR /dMMR” “MSI-High” “PD-1/PD-L1″ TMB”
There is also another new and exciting /emerging immunotherapy called “CAR T Cell”

The Immune systems basic process
  1. Identify – APC (Antigen Presenting Cells) are the immune system messengers. Their job is to seek out cancers, grab a piece of the suspicious cell, and deliver it back to the T Cells. This excites the T Cells.
  2. Activate – CTLA-4 (Check Point Protein) sits on the T Cells surface. Its job is to activate – Switch ON a T Cell attack based on the APC’s findings.
  3. Infiltrate – PD-1: (Programmed Death 1) Another Check Point Protein sits on the T Cells surface. Its job is to deactivate – Switch OFF a T Cell attack based on binding with a friendly PD-L1 on the surface of the suspicious cell, thus preventing healthy cells from being destroyed.

Our Immune System is very clever it has B Cells
It has an army of cells to remember encounters with threats – cells that remember our flu viruses also remember cancers.

Once a cancer is discovered, the B-Cell swing into action by searching within its ranks for one cell the can engage and learn this new threat. When one of the B Cell’s learns the DNA fingerprint of this new threat, it begins to replicate itself into the thousand who now know this cancer.

The B Cell replication creates 2 types of new B-Cells 

  1. ‘Memory B cells’ that remember the cancer
  2. ‘Effector B cells.’ These Effector B-Cells – antibody factories that produce millions of more B-Cell antibodies should the cancer ever return.

This is the huge upside to Check Point Inhibitors that not only keeps the T Cell’s attack switch in the ON position, but it also allows the B Cells to remember, thus one the immunotherapy is ceased after 2 years the B Cells will take over from there.

APC Explanation Video Resource 

The image above shows a tumour cell expressing a PD-L1 ligand and attempting to bind with the T Cells, PD-1 (Check Point Protein) A successful binding will result in the PD-1 switching off the T Cell attack. The image also shows the presence of the “Checkpoint inhibitor drug” Keytruda intercepting and blocking this binding /connection.

What are Immune Response T Cells

T Cells are relentless and very efficient killing machines, but they need to see the threat. When the immune system activates its T Cell army against a new threat, the activated T Cells express a checkpoint protein called PD-1 onto their surface

What are PD-1’s

PD-1 is an Immune inhibitory checkpoint molecule that is expressed by an ”Activated” T Cell. The PD-1 function is to bind and communicate with PDL-1 receptors that sit on the surface of healthy cells. When PD-1 successfully binds with a PD-L1 ligand-protein, it then transmits an inhibitory signal back to the T Cell to cease the attack.

What is PD-L1

PD-L1 Ligand is an elongated cluster protein that a cell expresses. PD-L1 ligands attach themselves to the membrane surface of a healthy cell. It’s function is to act as an immune regulatory molecule (A Check Point Molecule) that protects healthy cells from being attacked by T Cells.

The immune System has Check Points

Our immune system is essentially in a permanent attack mode and has inbuilt check points (PD-1) to switch off attacks when encountering healthy Cells. Tumours have learnt to exploit this by expressing the PD-L1 ligand on their cell surface.

Microsatellite Instability (MSI)

Word terms

  1. Microsatellites are parcels of DNA code
  2. Microsatellite Instability in those parcels (MSI)
  3. Mismatch Repair (MMR) our body’s DNA spell checker
  4. Deficient Mismatch repair (dMMR) means failure of the spell checker

Microsatellite Instability occurs when the strings of DNA contained within the microsatellites, replicate incorrectly creating errors in our DNA code.

MSI is described as follows
  1. MSI-high: (MSI-H) occurs if two or more microsatellite markers show instability
  2. MSI-low: (MSI-L) occurs if there is only one marker unstable
  3. MSI-Stable : (MSS) results when all the five microsatellite markers are stable
Reporting often describes MSI as follows
  1. MSI-h > 30%
  2. MSI-L <30%
  3. MSS – 0% instability
MMR the Spell Checker
  1. MMR – means all genes are functioning / expressing correctly
  2. dMMR – when one or more genes are not expressing and correctly

There are 4 primary MMR genes are MLH1, MSH2, MSH6, and PMS2
Think of these genes as our body’s spell checkers, their job is to express proteins that fix/repair DNA replication errors as they occur. This is a normal function within our body.

What is spell checking?
  1. Errors occur often in our DNA replication process.
  2. Microsatellites are parcels that contain strings of our pre-made dna code – the code that makes us – us!
  3. This replication process is constant and errors are normal
  4. When all 4 MMR genes are expressing proteins (spell checkers), then this is described as MMS (Stable).
When one or more of these genes fail to express their proteins (spell checkers) this means the MMR process is now deficient and is not correcting all DNA mistakes as they occur. This then leads to the accumulation of DNA replication errors within the microsatellites creating microsatellite instability.

Uncorrected errors lead to accumulations of defective cells that cluster and can form cancer mutations.

Immunotherapy and MSi-High
The MSi-high status has so far proven to perform best with Checkpoint Inhibitor Immunotherapy treatments, so this is the primary testing objective.

Testing for MSi-high

There are 2 primary testing methods to determine this, both require a tissue sample (biopsy)

  1. IHC fast efficient and inexpensive
  2. Molecular profiling more expensive and slower
  3. Blood Profiling – Still in early stage

IHC and Molecular test accuracies a 92% and 95% with blood much lower at this stage (2019)
For the purpose of discovering if you are a match for Check Point Immunotherapy, the IHC test is most logical. If via the IHC test you do not have the MSi-high as a biomarker then pursue the full “Molecular Profiling” to discover a full road map of your tumors make up and what mutations are driving your cancer. This can then be matched up with known treatments globally.

IHC Test Description

Immunohistochemistry Test – fast and Efficient  (3 -5 days) see Testing  Page 
What you testing for is MSi-high result, as immunotherapy responds well to this.

What do Microsatellites do?

They are the containers that hold our repetitive sequences of nucleotides (DNA code).

What are Nucleotides?
  1. Nucleotides are sequences of our DNA code. (Sequence repeats)
  2. Nucleotides are our “DNA Building Blocks”
  3. Nucleotides typically have 2 to 7 different types of pre programmed sequences of repeat code –
  4. Each sequence can contain between 2 -25 characters (string or stretch of code) – see diagram below.
  5. Nucleotide sequences continually replicate.
  6. Everyone has different pre programmed sequences.There can be hundreds of different code sequences.
The Nucleotide replication process can often go wrong

The MMR expressed proteins correct these spontaneous errors as they occur. When our MMR spell Checker is compromised or weakened then inconsistencies in the Microsatellites Nucleotides repeats over and over, this is when cancers can form.

Lab testing  (IHC) can easily discover this when comparing healthy cell numbers or sequences, with cancer cells. If a difference is identified, then this is referred to as “microsatellite instability”.

MSI levels/identification
Microsatellite Markers

Samples of Nucleotide code sequences contained within a microsatellite are supposed to replicate exactly the same in tandem.

Scott Paulson MD

Extract from CURE who spoke with Scott Paulson, M.D. medical oncologist with Texas Oncology, an affiliate of The US Oncology Network, to break down exactly what MSI-H status is, and the effects it may have on certain malignancies. Full Article link 

MSI-high tumours form a well-defined group with distinct clinicopathologic features (more easily identifiable) which characterizes an overall better long-term prognosis.

MSI-H is a feature of cancer’s genetic coding, which results in it behaving and “looking” a certain way on a microscopic level. Due to defects in the way that DNA in the cancer cells repairs itself, it creates changes and mutations to normal body cells that can eventually let them turn into cancer. However, these cells become so abnormal, because of this feature, that the immune system, which is used to protecting the body against bacteria and viruses and other foreign invaders, can actually look at the cancer and recognize that something is very wrong. This can call the body’s normal defenses against invaders to try to attack the cancer.

TMB is also a factor
Something to be aware of.

When the body has a dMMR/MSI-High environment it typically sees a higher TMB (tumour mutation burden) in the body and these tumours also have a strong correlation to higher expression of the protein-ligand PD-L1 which anchors to the surface of the tumour cells.

Tumor Mutation Burden (TMB) refers to the total amount of cancer tissue in the body. The survival of a patient relates to the tumor mutation burden, disease location and most importantly, the pace of the disease. In general, patients with a high burden of tumors have a high pace of disease and therefore a very short survival without therapy.

More About TMB

Source: Onclive 2018

Patients most likely to respond to immune checkpoint inhibitors (ICIs), tumor mutational burden (TMB) has emerged as a highly promising and clinically validated biomarker. Results from studies have demonstrated that subsets of patients with high TMB exist across almost all cancer types and that assessing TMB through whole-exome sequencing (WES) or next-generation sequencing (NGS) can predict response to a range of different types of immunotherapy.

TMB has been a hot topic at oncology conferences this year. Investigators and test developers must overcome a variety of challenges before it is ready for prime time as a biomarker, but it appears poised to bring immunotherapy into the era of personalized medicine.
A Heavy Load

It is a central tenet of cancer biology that tumors arise and evolve as a result of the acquisition of damage to the genome, generating characteristic genomic alterations that lead to the dysregulation of key cellular processes termed cancer hallmarks. In addition to identifying individual frequently altered genes that function as drivers of particular cancer types, results from genome sequencing studies have revealed the global spectrum of somatic mutations across a given tumor, known as the TMB or mutational load. TMB is defined as the number of mutations per megabase (Mb) of DNA.

Seeds of Their Own Destruction

It has long been suspected that cancers with a greater number of gene mutations may provoke a stronger antitumor immune response. The thinking behind this hypothesis relates to the production of neoantigens—fragments of proteins expressed on the surface of cancer cells that are encoded by mutated genes. Neoantigens are unique to cancer cells because they are derived from a mutant gene, which may encode a mutant protein that differs from that expressed by normal cells. Therefore, neoantigens have the potential to be recognized as foreign by the cells of the immune system that patrol the body. A greater number of neoantigens might mean increased stimulation of those immune cells and stronger immune response. In this way, cancer cells may generate the seeds of their own destruction.

My Kingdom for a Biomarker

MSI and dMMR and are just 2 of the many phenotypes that can cause hypermutant tumors and contribute to high levels of TMB. If tumors with higher TMB provoke a stronger immune response, then it stands to reason that TMB could provide a means for more comprehensive assessment of patients who might respond to ICIs and potentially other immunotherapies. In the past several years, investigators have started to evaluate TMB in this capacity, and it has emerged as a powerful predictor of response to ICIs in a range of tumor types Investigators are seeking to correlate TMB with clinical and biological outcomes in a number of ongoing clinical trials.

High TMB

Source: Caris life Sciences

Tumor Mutational Burden (TMB) is an emerging, quantitative indicator for predicting response to novel immune checkpoint inhibitors across a wide spectrum of tumor types. TMB measures the total number of non-synonymous, somatic mutations identified per megabase of the genome coding area (a megabase is 1,000,000 DNA base pairs).

Tumors with high TMB likely harbor neoantigens and may respond more favorably to immune checkpoint inhibitors. Caris Molecular Intelligence defines TMB as 17 or more mutations per megabase. TMB is included for all MI Profile orders, at no additional cost, added tissue, or delay in turnaround time.

How Tumor Mutational Burden Works
  • Non-synonymous mutations are changes in DNA that result in amino acid changes in the protein.
  • The new protein changes result in new shapes (neo-antigens) that are considered to be foreign to the immune system.
  • Immune checkpoint inhibitors are able to stimulate and allow the immune system to detect these neoantigens and destroy the tumor.
  • Germline (inherited) mutations are not included in TMB because the immune system has a higher likelihood of recognizing these alterations as normal.

A different approach, bypassing the Check Point Inhibitor.

What is the role of T-cells in the immune system?

T Cells are the soldiers of the immune system who search out and destroy foreign invaders, such as flu and cancer.

Chimeric antigen receptor CAR T Cell
  • CAR T Cell Therapy is a way of engineering the body’s T Cells to recognise cancer, by reprogramming the body’s own T Cells to recognise and destroy the cancer.
  • T Cells are removed from the patient and then the patient’s Tumour antigens are added to teach the T Cells.
  • The newly modified T Cells are then grown into the millions and then reintroduced into the patient’s body.
  • CAR T-cell therapy, is a new form of immunotherapy that uses specially altered T cells to directly and precisely target cancer cells.

The immune system is made up of a variety of cells and organs that normally protect the body from infection and cancer. An important component of the immune system is T cells, which have the capacity to hunt down and destroy abnormal cells, including some cancer cells.

Sometimes, cancer cells find ways to evade the immune system; so, the immune system needs to be retrained and enhanced to recognize and attack cancer cells. CAR T-cell therapy is one innovative approach to program and strengthens the immune system to attack some forms of cancer.

After a small portion of a patient’s own T cells has been collected from the blood, these cells are re-engineered in a special laboratory so they now carry special structures called chimeric antigen receptors (CARs) on their surface.

When these CAR T cells are reinjected into the patient, they multiply rapidly and these engineered receptors may help the T cells to identify and attack cancer cells throughout the body.

CAR T-cell therapy has been shown to be effective in B-cell acute lymphoblastic leukemia (ALL) and adult diffuse large B-cell lymphoma (DLBCL).

There are no approved CAR-T treatments worldwide for solid cancers. However, research is underway at Peter Mac in CAR-T cell treatment of solid cancers.  Please see Clinical for further information.

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Terminologies in Immuno 101

APC: Antigen Presenting Cells: The immunes messengers.
B Cells: learn the tumour or virus DNA to recognise if they return.
Biopsy: A tissue sample extracted from your tumor and used by a laboratory to discover Biomarkers.
CTLA-4: A Check Point protein that sits on the surface of the T Cells. They prevent mistake attacks on healthy cells.
Check Point Inhibitor– See PD-1 below
Car T Cells: An new innovating immunotherapy technique
IHC: Immunohistochemistry Test for discovering PD-L1 and MSi status
Ligand: An elongated strand of protein receptor that anchors to the cells surface. (ie PD-L1 is a Ligand receptor)
Molecular Profiling: Provides a DNA road map of the tumours fingerprint (Genomic) and your hereditary fingerprint (Genetic)
Microsatellites: Microsatellites are stretches of DNA that contain a repetitive sequence of nucleotides
Nucleotides: Nucleotides are the repetitive strings of DNA code that make us – us. example of a code  string;  “AAAAA or CGCGCGCG”  (codes are short tandem sequences that replicate)
MSI: is a measure of Microsatellite Instability
MSI-LOW: is a low recorded measure of Microsatellite Instability.
MSI-HIGH: is a high recorded measure of Microsatellite Instability.
MSS: is  Microsatellite Stable, which means no instability is present
MMR: Mismatch Repair is the DNA repair pathway that plays a key role in maintaining our genomic stability. MMR is our “Spell Checker” correcting any errors in our DNA replication process as they occur. MMR is made up of 4 proteins (MLH1, MSH2, MSH6, & PMS2 )
dMMR:  Deficient Mismatch Repair means that one or more of the 4 MMR proteins absent and as a result, the MMR is not functioning correctly and is described as deficient
TMB: means Tumour Mutation Burden
PD-L1: means Programmed Death Ligand 1 – a cluster protein that generally coats and protects healthy cells from the immune systems T Cells
PD-1: is an Immune Check Point Inhibitor – its function is to switch off a T Cell attack. It does by binding and communicating with the PD-L1 anchored to the surface of a cell.
PD1/ PD-L Pathway: A communication pathway/channel that dampens or deactivates (switches off) an immune response – (T Cell attack) on healthy cells.


Simple explanations and Video

Molecular Profiling & Videos

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