KW seminar: Principles of immunotherapy for head and neck cancer Flashcards
What are the study goals of this lectuer
(obv don’t study)
- Understand which steps are required to generate an effective anti-tumor immune response and know which cells are involved.
- Comprehend how T cells become activated and which signals are involved.
- Know what a tumor antigen is, how tumor cells can escape from immune recognition and elimination (3 E’s) and which mechanisms head and neck cancers use.
- Be able to explain the differences between various tumor microenvironments (TMEs) (immune inflamed, excluded, desert).
- Know which receptors on T cells are stimulatory and which are inhibitory for T cell functioning.
- Know the target and working mechanism for the following therapeutic antibodies (targeted antibodies or immune checkpoint inhibitors): cetuximab, nivolumab, ipilimumab, pembrolizumab.
- Know what head and neck cancer is, what the risk factors are for developing head and neck cancer, know the anatomical sites where they develop and know the different etiologies of head and neck cancer we can discriminate.
- Know which types of immunotherapies are being used in the clinic for the treatment of head and neck cancers.
- Know which resistance mechanisms can be present (intrinsic) or are developed (acquired) in tumor cells to counteract immune attack.
- Understand how the expression of PD-L1 in tumor tissue is being determined and quantitated as a biomarker in the clinical setting (CPS, TPS).
What is the outline of this lecture?
(obv don’t study)
- The immune system and cancer: basis for immune checkpoint inhibitors
- Head and neck cancer: development, complexity and treatment
- PD-L1 as a biomarker for anti-PD-(L)1 treatment in HNSCC
- The tumor microenvironment and resistance mechanisms to immunotherapy
What does the Immunoediting theory (aka 3E’s hypothesis) mean?
Immunoediting is a dynamic process that consists of immunosurveillance and tumor progression. It describes the relation between the tumor cells and the immune system. It is made up of three phases: elimination, equilibrium, and escape
Immunoediting is a theory that describes the transformation of normal cells to clinically-detectable cancer. The theory implies that while the human immune system protects from cancer, it also drives the development of tumors that will undergo immunogenic “sculpting/shaping” and may survive immune cell attacks.
Where do the three E’s in the 3E’s hypothesis stand for?
Elimination, equilibrium and escape, these are the 3 phases of immunoediting
Can you explain what happens in this figure?
(Since you already had this in detail in chapter 12/13, there is only one card about it)
Normal cells are exposed to carcinogens, radiation, chronic inflammation or viruses and therefore alter (top right in the figure). Gene expression is altered (e.g. reduced expression of tumor suppressor genes or upregulation of oncogenes). Because they start to invade the normal tissue, they will induce tissue damage that induces ‘danger signals’ (uric acid and extracellular matrix). In a normal situation, the ‘danger signals’ attract both the innate as well as the adaptive immune system (see second picture under ‘elimination). The transformed cells are removed by the immune system. However, there might be some cancer cells that are not detected or are transformed because of the immune response, which are not eliminated. At this point an equilibrium between the immune and cancer cells occurs (middle figure). However, due to genetic instability or immune selection this equilibrium can alter, upon which tumor heterogeneity occurs. When this situation is not handled and further evolves, the tumor will have certain ‘tricks’ to evade/actively suppress the immune system (e.g. down regulating MHC reduce responsiveness to IFNy, expressing IDO to actively suppress effector cells etc) (figure under ‘escape’)
What are the 7 steps of the cancer immunity cycle?
- Release of cancer cell antigens
- Cancer antigen presentation
- priming and activation
- Trafficking of T cells to tumors
- Infiltration of T cells into tumors
- Recognition of cancer cells by T cells
- Killing of cancer cells
Note: this cycle focuses mostly on T-cells
How can the cancer immunity cycle be used in the clinic?
It can be used as a guidance to determine at what step the cycle is malfunctioning and can provide clues as to what therapy might be a solution.
Can you place the following cells in the correct place (: innate or adaptive immune system): B cell, CD4+ T cell, CD8+ T cell, Dendritic cell, Granulocyte, Macrophage, Mast cell, Natural killer cell, Natural killer T cell, δɣ T cell, antibodies
You don’t have to learn this by heart, just a quick recap for you to think about
What is a tumor antigen? (+ name examples)
Tumor antigen is an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are useful tumor markers in identifying tumor cells with diagnostic tests and are potential candidates for use in cancer therapy. The field of cancer immunology studies such topics.
They can be identified because they are e.g. overexpressed, they expressed tissue-specific, they have a certain mutation (= different protein), they are specific for an oncogenetic virus or they have a cancer-germline gene that is different from the normal cells (see figure, it will make sense)
There is a correlation with the amount of somatic mutations and certain cancer types. Which cancer types have the highest amounts of somatic mutations?
Don’t learn by heart, get a feel for it :)
The most prevalent are melanoma, lung, stomach, esophagus, colorectum, bladder, uterus, cervix, liver and head- and neck cancer
What happens when there is co-stimulation (MHC-molecule + CD28 molecule) between an APC and T-cell?
There is T-cell activation (and it differentiates)
Note how the MHC binds to TCR and B7 binds to CD28
When there is T-cell activation (through co-stimulation), by natural processes CTLA4 is upregulated. What happens because of this?
CTLA4 has a higher binding affinity for B7 (on APC) than CD28, thereby blocking the co-stimulation. The T-cell is not activated and there is no differentiation.
Why is it important that the T-cell blocks the activation by means of upregulating CTLA-4?
Because if this doesn’t happen, there’s unlimited T-cell proliferation, leading up to many inflammatory diseases
How can the process of negative signaling from CTLA-4 (blocking T-cell activation) be used in the oncology department? (+ also name the drug)
You want the T-cell activation to last longer so that all cancer cells are killed. You can do this by blocking the CTLA-4 activation. The drug that was designed to do this is called ipilimumab, which binds to CTLA-4.
This is an example of immunotherapy
Explain how a tumor cell uses PD-L1 in it’s advantage
This slide did not have an explanation mark (I don’t know if this means it’s not, or if it’s less relevant)
The tumor cell (over)expressees PD-L1. PD-L1 binds to PD-1 and inhibits the T cell from killing the tumor cell.
(As discussed earlier, PD-1 is important in preventing autoimmunity by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity)
How can the expression of PD-L1 by tumor cells be used in the clinic? (+ what are the names of the drugs)
This slide did not have an explanation mark (I don’t know if this means it’s not, or if it’s less relevant)
By creating a drug that binds to the receptor (PD-1) or to the ligand (PD-L1). In that way, the function of the T-cell is not inhibited, and cancer cells are killed. Nivolumab and pembrolizumab are the two drugs used today.
Note: this is a form of immunotherapy
It has recently (2020) been discovered that inhibition of PD-L1 plays a double role. Explain these roles.
This slide did not have an explanation mark (I don’t know if this means it’s not, or if it’s less relevant)
PD-L1 inhibition acts both at the tumor-draining lymph nodes(tdLNS), and at thetumor site.
Lymph nodes that lie immediately downstream of tumors (tumor-draining lymph nodes (TDLNs)) undergo profound alterations due to the presence of the upstream tumor. The antigen-presenting cell population in TDLNs becomes modified such that tumor-derived antigens are cross-presented by host cells in a tolerizing fashion.
Texpng = progenitor T-cell
What does this picture show (also think about the relevance of tumor cells in this picture)?
(left is APC cell and/or tissues, right is a T-cell)
- T cell activation is controlled through a balans between activating and inhibitory co-receptors
- Tumors have a benefit when they are able to promote the expression of inhibitory receptors and ligands on their surface as well as the surface of surrounding (immune) cells
This picture shows T-cell stimulatory (green) and inhibitory (red) co-receptors
What is meant by the term ‘tumor microenvironment’?
The tumor microenvironment (TME) is the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix (ECM). The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells.
CTL: cytotoxic T lymphocyte, NK: natural killer, MDSC: myeloid-derived suppressor cell, TAM: tumor-associated macrophage, TCR: T cell receptor, Treg: regulatory T cell,
As stated earlier, tumors can influence the microenvironment. The type of tumor microenvironment can either be inflammatory or immune-suppressive. How is this applicable in the clinic?
The type of TME can determine prognosis and response to therapy
Where do Head and Neck squamous cell carcinoma’s (HNSCC) originate from? Can you also name their subtypes?
They originate in the epithelial layers of the upper aerodigestive tract. The subtypes are oral cavity and lip, laryngeal, nasopharyngeal, oropharyngeal and hypopharyngeal
Although it is stated as one type of cancer, as you can see there are many different types
What are the risk factors for Head and Neck squamous cell carcinoma’s (HNSCC)?
- Smoking, excessive alcohol use, beetle-nut chewing
- High risk HPV infection (HPV-16 most common)
- Genetic predisposition (Fanconi Anemia)
What is the incidence and mortality rate of Head and Neck squamous cell carcinoma’s (HNSCC)?
This is just as an indication, don’t learn please :)
- 3-5% of total cancer incidence
- incidence/year
- worldwide: >700,000 new patients
- Netherlands: ~3,000 new patients
- 40-50% of patients dies within 5 year
What are factors that will likely influence the tumor microenvironment, and immune suppressive pathways in HNSCCs?
- Etiology
- Molecular profile
- Localization within the upper aerodigestive tract
- Oral microbiome
Earlier, we noted that there are three different types of HNSCC: HPV-positive, HPV-negative with CNA-high tumors and HPV-negative with CNA-silent tumors. When looking at this figure, can you explain what happens with HPV-positive tumors?
This slide did not have an exclamation mark
We start off in the middle, with the normal mucosal epithelium. When looking at the HPV-positive tumors, we follow the arrow going to the left. Usually an HPV-infection does not cause any harm because the body gets rid of it. However, particular harmful HPV’s (like HPV-16) can cause chronic inflammtion, causing abnormal cells. Two different HPV-positive cells have recently been identified: HPV-IMU and HPV-KRT.
IMU seems to have a more active immune response than KRT, but more research is needed since the differences are still unknown between the two
Earlier, we noted that there are three different types of HNSCC: HPV-positive, HPV-negative with CNA-high tumors and HPV-negative with CNA-silent tumors. When looking at this figure, can you explain what happens with HPV-negative, CNA-high tumors?
This slide did not have an exclamation mark
Again, we start in the middle with normal epithelium. Due to carcinogens such as smoking, mutations can occur. Mutations that often happen are TP53 and CDKN2A, causing a ‘field cancerization’ (middle, bottom figure). These cells have a high Copy Number Alteration, meaning that there are lots of mutations (seen by the fluctuating graph). Also FAT1 and NOTCH1 are often mutated. There are different subtypes based on their mutation (e.g. mesenchymal, classical or basal)
Earlier, we noted that there are three different types of HNSCC: HPV-positive, HPV-negative with CNA-high tumors and HPV-negative with CNA-silent tumors. When looking at this figure, can you explain what happens with HPV-negative, CNA-silent tumors?
This slide did not have an exclamation mark
By unkown mechanisms (maybe age?) normal epithelial cells can be mutated. The genes that are often identified are HRAS and CASP8. There are not a lot of mutations (CNA-silent) as indicated by the flat line in the figure (top right).
What does this Kaplan-Meier curve show?
It shows the overal survival during a certain period. It shows that patients who are HPV-positive but have expression of p16, have a much better outcome after five years (~80%), than patients who are HPV-negative and also don’t have expression of p16 (less than 50%).
What does this Kaplan-Meier curve show?
That the prognosis in a later stage of HNSCC is very poor. Even if you have HPV-positive HNSCC, if your tumor is very malignant, the proganosis is poor so better treatment is needed.
Which has a better prognosis, CNA-high or CNA-silent tumors in HNSCC?
CNA-silent tumors, as indicated by this figure.
We don’t know why yet
An overview of the different treatments for HNSCC are given in this table
Interesting to know, but not necessary for the exam
Notice how we now discussed the three treatments in this table (Bleomycin is chemotherapy)
Briefly explain how Cetuximab (EGFR-inhibitor) works in HNSCC
The EGFR-pathway is often overexpressed, causing cell proliferation, growh, angiogenesis, survival and metastasis. Cetuximab binds to the EGFR receptor, preventing the (downstream) activation and signaling. Thus, the proliferation and growth is inhibited.
Besides inhibiting EGFR signaling, Cetuximab also has an immune-related mode-of-action called: antibody-dependent cellular cytotoxicity. How does this work?
While cetuximab is bound to the EGFR receptor on the tumor cell, the tail can be recognized by the CD16/FcyRIII receptor on NK-cells. This causes activation of the NK-cell, which releases Perforin and granzyme B, that kill the tumor cell. After being killed, the tumor antigens are released and recognized by dendritic cells, which then further activate T cells.
Unfortunately, resistance in the mechansims of cetuximab-induced ADCC (antibody-dependent cellular cytotoxicity) is seen. How does this resistance work?
- NK-mediated cytotoxicity is inhibited by the interaction of PD-L1 on the tumor and PD-1 on the activated NK cell
- The presence of HLA class I / MHC I on tumor cells blocks NK cell cytotoxicity via interaction with inhibiting KIR receptors
This can be seen by the red arrows in the figure. Because the tumor cell is not inhibited, the tumor growth further
Why are we so enthousiastic about (personalized) immune therapy?
When looking at the left curve, showing the tumor volume, in both chemo- as well as targeted therapy, the survival time is elonged, but eventually the tumor catches up and the patients die (steep curve). In immune therapy we see that after an amount of time the tumor growth is not only inhibited, but further reduced. Also if we look at the survival rate, we see that cancer becomes a ‘chronic’ rather than a ‘terminal’ disease
Note that personalized immune therapy is (currenly) only available in a small group of patients
What were the results for Nivolumab in HNSCC during the 2nd line treatment?
Obv don’t learn by heart, just for indication
This figure shows recurrent/metastatic HNSCC progressed during or within 6 months after platinum-containing chemotherapy (so chemotherapy did not work anymore)
- Arm 1: nivolumab (3 mg/kg every 3 wk)
- Arm 2: single agent standard care (methotrexate, docetaxel or cetuximab)
After this experiment Nivolumab was approved for HNSCC
What were the results of Pembrolizumab in HNSCC (2nd line treatment)
As you can see, there is not a huge difference between HPV-positive and HPV-negative (24% vs 16%). When the tumor growth was 30% decreased, this was seen as a ‘response’ and thus 18% of the patients had an overall response rate. Note that only PD-L1 tumors are used in this trial, and that there is no control group and/or RCT
What is the immune serveillance in HNSCC?
(6 steps, both positive and negatvie)
Positive actions by immune cells, shown in green (the numbers correlate with the numbers in the figure)
- Uptake of tumor antigens by APC in the mucosa
- Migration of APCs to the lymph nodes (LNs)
- Cross-presentation of tumor antigens to T cells in the LNs; T cell priming
- Migration of T cells to the tumor and tumor cell killing
Negative (inhibitory) actions by tumor cells, shown in orange and red
- Inhibition of APC activation and migration (a) and altering myelopoiese in the bone marrow (b) by tumor-derived factors (TDF)
- Inhibition of immune surveilance by TDF-induced myeloid derived suppressor cells (MDSC) and Treg, by blocking the functions of APC and T cells
What are some different mechanisms of immune escape in HNSCC?
- Development of T-cell tolerance to persistent HPV infection, overexpressed/muated antigens
- Production of low genome copy numbers in the basal layer of th epithelium
- Increased PD-L1 expression in HPV-positive tumors and increased PD-1 expression in cytotoxic T lymphnocytes
- Downregulation of interferon regulatory factors and activated STAT1
- Inhibition of inflammatory cytokines and transcription factors
- Downregulation or mutation of HLA class I and antigen-processing machinery components
I marked the mechanisms that were not discussed yet, the other you should recognize
CPS (: combined positive score) is a method to see when is a tumor PD-L1 positive. How does this work?
The percentage of PD-L1 expression on tumor cells (partial or complete membranous staining) and membrane and/or cytoplasmatic expression of PD-L1 on infiltrating immune cells (lymphocytes en macrophages) in the tumor or adjacent tumor stroma as a relative score in relation to the total number of viable tumor cells
Can you give an CPS calculation example?
THIS IS FOR YOUR OWN UNDERSTANDING
For example, a tumor is shown where 10% shows a PD-L1 positive area. A pathologist will count 100 cells, and will count which of those are PD-L1 positive. In this example 80% of the cells are PD-L1. However, this is only 10% of the tumor area, so there is a CPS of ~8.
You could also divide the specimin into quarters, and then count all the cells per quarter and take the average to asses the total CPS of the tumor
This figure shows on the left PD-L1 expressors, and on the right PD-L1 non-expressors. The response of nivolumab in HNSCC (with regard to PD-L1 expression) was investigated. This PD-L1 expression was determined based on the TPS (tumor proportion score). When comparing the two figures, there is no clear distinction made between the two groups. Can you explain this phenomenen?
This can be explained by tumor heterogeneity. As can be seen in this figure, many slices were made of an autopsy. If you had a slice that is shown by the red square, this means the patient is PD-L1. However, all the other slices did not show PD-L1 positivity (while the patient is clearly PD-L1 positive). Therefore, PD-L1 cannot be used as a biomarker, due to the tumor heterogeneity
The tumor immunity continuum can be divided into three categories (based on T-cells): inflamed, immune excluded and immune desert. Can you explain what each means and by which (tumor) gene expression they are caused?
-
Inflamed: immun cells are infiltrated in the tumor, but inhibited
- By INFy-signal expression
-
Immune excluded: CD8+ T cells are accumulated but have not efficiently infiltrated the tumor (are at the border)
- By TGFß-signal expression
-
Immune desert: CD8+ T cells are absent from tumor and its periphery
- By fatty acid metabolism, neuroendocrine features, and the Wnt/ß-catenin pathway
By which tumor immunity continuum (inflamed, immune excluded or immune desert), do you expect to be a response by immunotherapy?
Inflamed, because the T cells are in the tumor, but need to (only) be activated. The immune excluded and desert tumors often don’t respond to treatment.
How does the Wnt/ß-catenin pathway play a role in immune-excluded/silent tumors?
If ß-Catenin is active, this will inhibit the expression of CCL4. CCL-4 is needed to attrackt cross-presenting dendritic cells to the tumor area. If there is no CCL-4, the macrophages can’t take up the tumor antigens and migrate to the lymph nodes to prime the T-cells. So there are no T-cells in the tumor when ß-Catenin is active.
What immune response is active during an inflamed tumor?
Because e.g. DNA is released, this is picked up by a dendritic cell, causing it to activate. However, at the same time, PD-L1 is activated by the tumor cell. The dendritic cell activates the ‘STING’ pathway, releasing type I IFN. This will result in priming and activation of T cells in the lymph nodes. Primed and activated T cells migrate to the tumor, and cytokines (CXCL9/10) are released. However, the tumor is expressing PD-L1, so it is not killed by the activated T-cells.
There are different resistanc mechanisms, which can be divided into intrinsic and extrinsic pathways. Can you name the processes per pathway (intrinsic: 4, extrinsic: 3)
I don’t think you supposed to know this by heart? Maybe try to recognize :)
Tumor cell intrinsic
- Absence of antigenic proteins
- Absence of antigen presentation
- Genetic T cell exclusion
- Insensibility to T cells
Tumor cell extrinsic
- Absence of T cells
- Inhibitory immune checkpoints
- Immunosuppressive cells
Please also read the ‘examples’ in the table
HPV+ and HPV- HNSCC have a different level of immune infiltrate. Fill in:
- T cell infiltration score in HPV-negative HNSCC is higher/lower than in HPV-positive HNSCC
- The total immune infiltration score in HPV-negative HNSCC is slightly higher/lower than in HPV-positive HNSCC
- T cell infiltration score in HPV-negative HNSCC is lower than in HPV-positive HNSCC
- The total immune infiltration score in HPV-negative HNSCC is slightly lower than in HPV-positive HNSCC (Note: they both score high!)
HNSCC have a different immune infiltrate dependent on etiology or site. However, a lot of studies nowadays still throw all HNSCCs on one pile. The lecturer thinks this has to change and wrote a review, where was shown that studies that had a negative effect on the growth of the tumor (= good treatment) were better when there was differentiation between the subtypes
this is just an info slide you can skip
- For HNSCC, the majority of studies report a favorable prognosis when high CD8+ T cell infiltrate is seen
- The presence of Treg is sometimes favorable, sometimes not
- Abundance of macrophages in the TME is related to an unfavorable prognosis in HNSCC
In the beginning of the lecture, we looked at the The Cancer Immunity Cycle (see figure). Can you name some therapeutic interventions based on the tumor profile? (3 answers)
- Reduce local immunosuppression in the tumor (e.g. oncolytic virusses, chemo- and/or radiotherapy) (1).
- Activate (or introduce by vaccination) dendritic cellc, to in vivo generate a T cell response (TLR- L/cytokine stimulation or DC vaccination) (2).
- (Re)activete the existing anti-tumor T cell response (Immune checkpoint inhibitors) (3,7) or provide tumor-specific T cells (CAR or TIL therapy, when immune-silent) (6).
