Theme 2

Why does cancer usually require multiple mutations?

Cells must acquire several properties, such as uncontrolled proliferation, resistance to cell death and immune evasion. One mutation usually cannot provide all these properties. A tumour can be benign or malignant; cancer is malignant.

How do CD8⁺ T cells and NK cells recognise tumour cells differently?

CD8⁺ T cells: recognise peptides presented on HLA-I through their TCR.
NK cells: respond to the balance of activating and inhibitory signals; stress ligands such as MICA/B activate NKG2D, while loss of HLA-I can remove inhibition.

How are intracellular tumour antigens presented to CD8⁺ T cells?

Proteins → proteasome generates peptides → TAP transports peptides into the ER → loading onto HLA-I → presentation at the cell surface. A mutation is only recognisable if its peptide passes these steps and binds the patient’s HLA

Why can high tumour mutation burden favour checkpoint inhibitor responses?

More mutations increase the chance of producing a presented, recognisable neoantigen. This does not guarantee a response. Melanoma often has high mutation burden; Lynch syndrome involves inherited mismatch-repair defects that can produce highly mutated tumours.

What does immune-cell infiltration tell us about tumour prognosis?

CD8⁺ T-cell infiltration generally predicts better prognosis, but cell type, location and cancer type matter. Tregs usually suppress immunity, yet can correlate with better prognosis in colorectal cancer; their presence may reflect an active immune response.

How do therapeutic cancer vaccines work, and which main platforms exist?

They deliver tumour antigens to APCs, especially dendritic cells, to activate tumour-reactive T cells. Platforms include cell-based, viral-vector, peptide and DNA/mRNA vaccines. Effective antigen delivery, APC activation and overcoming suppression are essential.

How do tumour-associated antigens differ from neoantigens?

tumour-associated antigens: self-antigens also expressed in some healthy tissues; potentially shared between patients, but subject to tolerance and healthy-tissue targeting.
Neoantigens: arise from tumour-specific mutations; often require personalised sequencing and production, increasing time and cost.

What is adoptive T-cell transfer, and how do TIL and blood-derived approaches differ?

T cells are collected, expanded and/or modified outside the body, then infused.
TIL therapy: expands T cells isolated from tumour tissue, usually followed by infusion after lymphodepletion.
Blood-derived approach: selects or expands tumour-reactive T cells from peripheral blood.

how do CAR T cells recognise cancer, and why does CD19 targeting affect healthy cells?

An engineered CAR directly recognises a surface antigen independently of HLA. CD19 occurs on malignant and normal B cells, so treatment can cause B-cell depletion. Target expression on healthy tissues determines potential toxicity.

What is the rationale and major limitation of IL-2 therapy?

IL-2 promotes T-cell proliferation and activation. It can produce durable responses in some patients with melanoma or renal cell carcinoma. Systemic administration causes substantial toxicity, especially capillary leak syndrome, hypotension and organ dysfunction.

How do oncolytic viruses stimulate antitumour immunity?

They preferentially replicate in tumour cells and cause lysis, releasing tumour antigens alongside viral danger signals. This promotes APC activation and immune responses. Viruses can also be engineered to express therapeutic molecules.

How does CTLA-4 inhibit T-cell activation?

CTLA-4 competes with CD28 for CD80/CD86 on APCs, reducing costimulation, particularly during T-cell priming. Blocking CTLA-4 increases activation. Example: ipilimumab.

How does the PD-1 pathway suppress antitumour T cells?

PD-1 on T cells binds PD-L1/PD-L2, inhibiting T-cell activity, particularly in peripheral tissues and tumours. Blockade can restore activity. Pembrolizumab: anti-PD-1. Atezolizumab: anti-PD-L1. Antigen recognition must still be possible.

what are the three phases of cancer immunoediting?

Elimination: immune cells destroy tumour cells.
Equilibrium: immunity restrains surviving cells while resistant variants are selected.
Escape: resistant or immunosuppressive variants grow into a clinically apparent tumour.

Which major mechanisms allow tumours to escape immune attack?

lose antigens; reduce HLA-I or antigen processing; lose activating stress ligands; increase inhibitory ligands such as PD-L1; resist apoptosis; create an immunosuppressive environment. Random variation followed by clonal selection favours resistant cells.

How can HLA-I loss help tumours escape, and what forms can it take?

Reduced HLA-I prevents CD8⁺ recognition. HLA-A, -B and -C can provide up to six different molecules. Loss may affect all HLA-I, one haplotype, one locus or one allele. Low HLA-I may activate NK cells, but retained inhibitory molecules such as HLA-E can limit this.

how do CD8⁺ T cells and NK cells kill targets, and how can tumours resist?

FasL–Fas: activates apoptotic signalling.
Perforin–granzyme: perforin enables granzyme entry; granzymes trigger cell death.
Perforin is a pore-forming protein, not a digestive enzyme. Tumours can reduce Fas or disrupt apoptotic pathways, including caspases.

How does the tumour microenvironment suppress immune cells?

Tumours recruit Tregs and MDSCs and promote suppressive myeloid cells. IL-10/TGF-β inhibit immunity; arginase/IDO reduce amino-acid availability. Competition for glucose and other nutrients also weakens T-cell function.

Why can IFN-γ have both antitumour and tumour-promoting effects?

Antitumour: increases antigen presentation and supports immune attack. Persistent signalling: can induce PD-L1 and IDO, promoting suppression and selecting resistant cells. Its effect depends on duration, signalling strength and tumour context.

What is the difference between primary and acquired resistance to immunotherapy?

primary: no effective response from the start. Acquired: initial response followed by progression. Resistant clones survive and expand through clonal selection, for example after antigen loss, defective presentation or increased immune suppression.

Which three factors favour a response to checkpoint blockade?

Earlier treatment: tumour antigens and immune structures are still present. More foreignness: mutations can generate recognisable neoantigens. More immune infiltration: tumour-reactive immune cells are available for activation.

What is the difference between pMMR/MSS and dMMR/MSI tumours?

pMMR/MSS: functional mismatch repair, generally fewer mutations and poorer immunotherapy responses. dMMR/MSI: defective mismatch repair → accumulated mutations → more potential neoantigens and often greater immune infiltration.

how common is dMMR in colorectal cancer?

Approximately 15% of non-metastatic and 5% of metastatic colorectal cancers. Most colorectal cancers are pMMR, not dMMR.

What is the key predictive biomarker for immunotherapy highlighted in this lecture?

MMR deficiency/MSI, which strongly predicts checkpoint inhibitor sensitivity. It occurs across different cancer types, although responses can differ by organ of origin.

Which checkpoint treatments are used for metastatic dMMR colorectal cancer?

Pembrolizumab (anti-PD-1) or nivolumab + ipilimumab (anti-PD-1 + anti-CTLA-4). These approaches can be given without chemotherapy.

Why combine checkpoint inhibitors?

they block different inhibitory pathways, potentially producing a stronger immune response. Dual blockade can improve response and survival in metastatic disease, but can also increase immune-related toxicity

How do neoadjuvant and adjuvant treatment differ?

Neoadjuvant: before surgery. Adjuvant: after surgery, targeting residual microscopic disease and reducing recurrence risk

What is the conventional treatment pathway for early-stage colon cancer?

surgery → pathological staging/risk assessment → adjuvant chemotherapy when indicated, especially with positive lymph nodes. The lecture reports 20–40% recurrence in high-risk disease despite surgery and chemotherapy.

Why might immunotherapy work particularly well before surgery?

the intact tumour provides abundant antigens, with tumour-reactive immune cells and draining lymph nodes still present. Treatment can expand systemic antitumour T cells and generate memory, potentially controlling micrometastases.

What were the key NICHE-2 results?

short-course neoadjuvant nivolumab + ipilimumab in dMMR colon cancer produced approximately 98% pathological response, 95% major pathological response and 68% complete pathological response. The lecture reports 100% 3-year DFS in the studied cohort.

What is the difference between pathological response, MPR and pCR?

pathological response: treatment-related reduction in viable tumour on tissue examination. MPR: ≤10% viable tumour remains. pCR: no viable tumour remains in the assessed surgical specimen, including assessed lymph nodes.

which alternative checkpoint combination showed high activity in dMMR colon cancer?

Nivolumab + relatlimab: anti-PD-1 + anti-LAG-3. The lecture reports approximately 97% pathological response and 69% pCR. This does not establish superiority over other regimens.

Is longer neoadjuvant immunotherapy always better, and can surgery always be omitted after a response?

Neither is established. Duration and single versus dual blockade remain open questions. Goals include resectability, long-term disease control and organ preservation. Clinical complete response does not automatically prove pathological complete response.

Can pMMR colon cancers respond to immunotherapy?

yes, a subset can, particularly in the neoadjuvant setting. The lecture reports responses in 8/31 patients (26%) after nivolumab/ipilimumab. pMMR means less responsive, not universally unresponsive.

Why does immune activation not necessarily mean tumour response, and how might pMMR responses improve?

Checkpoint blockade activated immunity in both responders and non-responders; activation alone is insufficient. Strategies aim to turn “cold” tumours “hot” through combinations with radiation, targeted therapies, chemotherapy or treatments that reduce immune suppression.

What are the main steps of the cancer–immunity cycle?

Antigen release → uptake and presentation by APCs → T-cell priming in draining lymph nodes → trafficking → tumour infiltration → cancer-cell recognition → killing, releasing more antigens. Failure at any step can prevent tumour control.

Why do APCs need danger signals?

PAMPs indicate microbes; DAMPs indicate cell stress or damage. They promote APC maturation, costimulation and effective T-cell priming. Antigen uptake alone may be insufficient

What causes dMMR colorectal cancer, and is it always hereditary?

Defects in MLH1, PMS2, MSH2 or MSH6 impair mismatch repair. Most cases are sporadic, commonly through MLH1 promoter methylation. Lynch syndrome involves inherited MMR defects and accounts for approximately 2–3% of all CRC.

Why are dMMR colorectal cancers generally more immunogenic than pMMR cancers?

dMMR produces more mutations and potential neoantigens, increasing the chance of T-cell recognition. However, mutation burden alone does not guarantee peptide presentation, recognition or treatment response.

In which CRC group was HLA-I loss particularly frequent in the presented research?

dMMR tumours, which face strong immune selection. Losing HLA-I allows escape from conventional CD8⁺ T cells despite abundant mutations. Your note saying mainly pMMR is reversed.

How do B2M mutations impair tumour recognition?

β2-microglobulin is essential for stable HLA-I surface expression. Loss-of-function mutations can prevent HLA-I presentation, making tumour cells invisible to conventional HLA-I-restricted CD8⁺ T cells.

How can defective IFN-γ signalling promote immune escape?

normally, IFN-γ increases antigen processing and HLA-I expression. Defects in pathways such as JAK–STAT can prevent this response, reducing tumour visibility and contributing to resistance.

What is the paradox of immunotherapy responses in HLA-I-deficient dMMR cancers?

Some respond well to checkpoint blockade despite lacking the HLA-I needed by conventional CD8⁺ T cells. This implicates additional effectors, including γδ T cells, that recognise targets independently of classical HLA-I.

Why might NK cells not fully compensate for tumour HLA-I loss

Loss of HLA-I removes inhibitory signals, but killing still depends on sufficient activation and tumour access. NK-cell infiltration can be limited, and PD-1 expression is less common, so anti-PD-1 does not directly activate all NK cells.

How do γδ T cells differ from conventional αβ T cells?

They express a γδ TCR and can recognise stress-related targets without classical peptide–HLA-I presentation. They also express innate receptors such as NKG2D, making them potential effectors against HLA-I-deficient tumours.

How do Vδ1 and Vδ2 T cells differ, and which subset was important here?

Vδ1: enriched in tissues, including the gut. Vδ2: prominent in blood, commonly paired with Vγ9. In the presented research, PD-1⁺ Vδ1 cells showed strong killing of HLA-I-negative CRC cells.

How do inhibitory KIRs differ from NKG2D

inhibitory KIRs recognise self HLA-I and restrain killing; HLA-I loss removes this brake. NKG2D is activating and recognises stress ligands such as MICA/B and ULBPs. They are different receptor systems.

Which other HLA-I-unrestricted cells were investigated besides γδ T cells?

ILC1-like cells, which were enriched in tumours and could recognise HLA-I-deficient targets. They were transcriptionally distinct from conventional NK cells.

How does the CD47–SIRPα pathway protect tumour cells?

CD47 on tumour cells binds SIRPα on macrophages, delivering a “don’t eat me” signal that inhibits phagocytosis. Immune escape therefore involves macrophages as well as T cells.

Why combine spatial transcriptomics with imaging mass cytometry?

Spatial transcriptomics: maps gene expression within tissue. Imaging mass cytometry: maps many proteins and cell types simultaneously. Together they reveal which cells are present, where they are and how they interact during tumour progression.

Do pMMR colorectal cancers lack all neoantigen-specific T cells?

No. The presented study detected neoantigen-specific responses in approximately 50% of the studied patients. Poor checkpoint responses therefore do not necessarily mean tumour-reactive T cells are absent.

Which markers can enrich for tumour-reactive CD8⁺ T cells?

Co-expression of CD39 and CD103 identifies a population enriched for tumour/neoantigen-reactive CD8⁺ T cells. This helps distinguish useful tumour-reactive cells from unrelated bystander T cells.

How could neoantigen-reactive T cells be exploited therapeutically?

Vaccination: stimulate them inside the patient. Adoptive transfer: isolate and expand reactive cells before reinfusion. TCR engineering: transfer a tumour-reactive TCR into T cells; conventional αβ TCR recognition remains HLA-dependent.

Why is the tumour microenvironment important in pancreatic ductal adenocarcinoma (PDAC)?

PDAC contains abundant stroma and suppressive immune populations, which can restrict effective immunity. The presented research showed that neoadjuvant treatment can alter this environment and increase T-cell infiltration. More PD-1⁺ cells do not automatically mean ineffective immunity.

What is immunogenic cell death, and why can it help cancer treatment?

Cell death that also stimulates immunity through antigen release and danger signals, such as ATP release, HMGB1 release and surface calreticulin. Some chemotherapy or radiotherapy regimens can induce it, helping APCs initiate antitumour responses.

What defines triple-negative breast cancer (TNBC)?

Absence of ER, PR and HER2 positivity. Therefore, endocrine therapy and conventional HER2-targeted therapy are ineffective. This does not mean that no targeted treatment options exist.

Is all breast cancer immunologically “cold”?

No. Immune infiltration varies. TNBC generally has more tumour-infiltrating lymphocytes (TILs) than ER⁺/HER2⁻ breast cancer. Lack of hormone receptors itself does not explain immune recognition.

What is the role of PD-L1 in metastatic TNBC treatment selection?

PD-L1 expression, assessed using a combined positive score (CPS), helps select patients for first-line pembrolizumab plus chemotherapy. CPS includes PD-L1-positive tumour cells and certain immune cells.

Why combine chemotherapy with checkpoint blockade?

Chemotherapy can kill tumour cells and modify the immune environment, increasing antigen release, inflammatory signalling and immune-cell recruitment. Its effects are drug-dependent: it can also cause immunosuppression.

Which five induction groups were studied in TONIC?

No induction, radiotherapy, cyclophosphamide, cisplatin or doxorubicin, followed by anti-PD-1 treatment.

Which immune changes followed cisplatin or doxorubicin induction?

increased T-cell infiltration, TCR clonality and inflammatory “hot” gene signatures. Greater clonality suggests expansion of selected T-cell clones; it does not alone prove tumour specificity.

What eosinophil change was associated with checkpoint responses?

Responders showed an increase in circulating eosinophils, associated with better outcomes. This patient association alone does not establish causation.

What evidence supported a functional role for eosinophils?

In mouse models, eosinophil depletion reduced the benefit of chemotherapy plus checkpoint blockade and impaired the associated CD8⁺ T-cell response. This supports a causal contribution in those models.

Are eosinophils always antitumour cells?

No. They can support tumour growth and a suppressive environment, or promote cytotoxicity and CD8⁺ T-cell recruitment/activation. Their role depends on context.

What different roles did IL-5 and IL-33 play in the proposed mechanism?

Checkpoint blockade → CD4⁺ T-cell IL-5 production → systemic eosinophil expansion. Adding cisplatin increased IL-33, promoting eosinophil infiltration into the tumour and supporting antitumour immunity.

Which factors predict prognosis in early-stage TNBC?

Tumour size, lymph-node involvement, TIL abundance and response to neoadjuvant treatment. Higher TIL levels generally predict better prognosis.

What is pCR, and why is it important in TNBC?

Pathological complete response: no residual invasive cancer in the breast or assessed lymph nodes after neoadjuvant treatment (ypT0/Tis ypN0). It is associated with better long-term outcomes, but does not guarantee cure.

What treatment strategy did KEYNOTE-522 test?

What were the main KEYNOTE-522 benefits shown in the lecture?

In high-risk early TNBC, neoadjuvant pembrolizumab plus chemotherapy, followed by surgery and adjuvant pembrolizumab, versus the same chemotherapy with placebo.

pCR: approximately 65% versus 51%. Five-year overall survival: approximately 87% versus 82%. The strategy improved both pathological response and survival.

does PD-L1 negativity exclude pembrolizumab benefit in early TNBC?

No. Increased pCR rates were observed in both PD-L1-positive and negative groups. Unlike metastatic TNBC, PD-L1 positivity is not required for the early high-risk treatment strategy.

Why is “only 15% benefit from anti-PD-1” misleading?

the approximately 14-percentage-point increase in pCR is the difference between treatment groups. It does not identify exactly which individuals benefited, nor capture every possible survival benefit.

Why is avoiding unnecessary immunotherapy important in potentially curable patients?

Checkpoint blockade can cause colitis, pneumonitis and endocrine damage, including thyroid dysfunction, adrenal insufficiency and diabetes. Some effects are permanent. Research asks who needs treatment and whether the full duration is necessary.

Why is the immune system’s role in cancer paradoxical?

It can eliminate cancer cells, but also promote tumour growth and metastasis through chronic inflammation, immune suppression and tissue remodelling. Its effect depends on cell type and context.

What does increased cancer risk in immunosuppressed patients demonstrate?

it supports a protective role for immune surveillance, particularly against virus-associated cancers. However, immunosuppression does not increase every cancer type equally.

How can chronic inflammation promote cancer development?

Persistent inflammation generates DNA-damaging molecules, growth signals and repeated tissue repair. Examples include inflammatory bowel disease → CRC, H. pylori gastritis → gastric cancer, and asbestos exposure → mesothelioma

Does adaptive immunity prevent all spontaneous tumours?

No. It protected against chemically induced tumours in some experiments, but removing T and B cells did not significantly change tumour onset in the presented mammary tumour models. Protection is context-dependent.

How do tumours build an immunosuppressive microenvironment?

tumour-derived signals recruit and alter neutrophils, macrophages and Tregs. Mediators such as TGF-β, IL-10 and PGE₂ suppress effective antitumour responses.

Can myeloid cells promote cancer without suppressing T cells?

Yes. They can also support angiogenesis, extracellular-matrix remodelling, invasion and metastatic niches. Their effects extend beyond immune suppression.

What is cancer-associated systemic inflammation?

Tumour-derived signals alter immune activity throughout the body, including bone-marrow myeloid-cell production and circulating immune cells. This can support tumour growth and distant metastasis.

What systemic neutrophil changes were observed in metastatic breast cancer?

Patients showed increased circulating neutrophils, altered neutrophil protein expression and increased nitric oxide production compared with healthy controls. The magnitude varied between patients.

Why are patient correlations insufficient to prove that neutrophils cause metastasis?

Neutrophils could be a consequence or marker of advanced disease. Functional experiments, such as cell depletion, are needed to test a causal contribution.

How did neutrophils from tumour-bearing mice affect CD8⁺ T cells?

They suppressed CD8⁺ T-cell proliferation. Increased iNOS/nitric oxide contributed to the suppressive programme described in the lecture.

What happened after neutrophil depletion in the presented mouse model?

anti-Ly6G reduced metastasis, while primary tumour growth changed little. This indicates that neutrophils particularly supported metastatic progression in that model.

What inflammatory cascade promoted breast cancer metastasis?

IL-1β → γδ T-cell IL-17 → G-CSF → neutrophil expansion and polarisation → suppression of CD8⁺ T cells → increased metastasis.

are γδ T cells always antitumour cells?

No. Some directly kill tumours, whereas IL-17-producing γδ T cells can promote neutrophil-driven inflammation and metastasis. Different subsets have different functions.

How did tumour p53 loss affect neutrophils?

p53 loss increased systemic neutrophil accumulation and pro-metastatic inflammation. It did not cause loss of neutrophils, as stated in your notes.

How did p53 loss connect tumour genetics to systemic inflammation?

p53 loss → increased tumour Wnt signalling → macrophage IL-1β production → γδ T-cell/neutrophil cascade → CD8⁺ suppression and metastasis.

Why do patients have different tumour immune landscapes?

Differences in tumour genetics, tissue context, disease stage, host factors and treatment shape immune-cell composition and function. The same cancer type can therefore contain very different immune environments.

Why should immune interventions be personalised?

Different tumours depend on different suppressive pathways. Matching treatment to tumour genetics and the immune landscape may improve efficacy and help convert tumour-supporting inflammation into antitumour immunity.

Why should we not simply eliminate all myeloid cells?

myeloid cells can also provide antigen presentation, pathogen defence and tumour killing. Treatment should target harmful populations or programmes while preserving useful immune functions.

What are the two main mechanisms of oncolytic viruses (OVs)?

Direct oncolysis: viral replication kills tumour cells. Antitumour immunity: released tumour antigens and viral danger signals activate immune responses, potentially attacking distant, uninfected tumour cells.

How can viruses naturally favour tumour cells?

Tumour conditions, such as defective antiviral responses and altered cellular signalling, can favour viral replication. This is natural tumour selectivity.

How can tumour selectivity be engineered?

Delete/mutate viral genes needed for replication in healthy cells, or place essential viral genes under tumour-selective promoters, such as E2F, nestin or survivin.

Why was human adenovirus serotype 5 useful for development?

it has a well-characterised, non-integrating dsDNA genome, permits high-titre production and is physically stable. Its human species specificity complicates testing in animal models.

What is Delta24-RGD, also called DNX-2401?

An engineered oncolytic adenovirus with two key modifications: a 24-base-pair deletion for selective replication and an RGD-4C insertion to improve infection.

How does the Delta24 deletion restrict viral replication?

it disrupts viral E1A binding to Rb. In healthy cells, functional Rb restrains E2F and cell-cycle entry. Tumours with a defective Rb pathway already provide conditions supporting viral replication.

What is the purpose of the RGD-4C insertion?

it enhances viral entry through integrins, improving infection of tumour cells with limited expression of the usual adenovirus receptor, CAR. Entry enhancement and replication selectivity are different functions.

Why is glioblastoma difficult to treat?

It shows diffuse infiltration into brain tissue, tumour heterogeneity and treatment resistance. These features limit complete surgical removal and durable disease control.

What preclinical efficacy evidence was needed before patient testing? glioblastoma

Demonstrate tumour-selective replication, killing of patient-derived GBM cells, activity in 3D organotypic cultures, and efficacy in relevant animal models.