theme 3

How are haematological malignancies broadly classified?

By cell lineage and maturation stage. Immature myeloid/lymphoid cells: AML/ALL. Myeloid neoplasms: CML, PV, ET and myelofibrosis. Mature lymphoid malignancies: CLL, lymphomas and plasma-cell myeloma.

Why does acute leukaemia cause fatigue, bleeding and infections?

Malignant blasts disrupt normal blood production → anaemia causes fatigue; thrombocytopenia causes bleeding; neutropenia causes infections. Total leukocytes may be high, normal or low. Blasts can enter blood before marrow is completely packed.

How is acute leukaemia diagnosed and classified?

Blood/bone-marrow morphology, flow cytometry to establish lineage, and cytogenetic/molecular testing to identify abnormalities guiding diagnosis, prognosis and treatment.

What is the difference between AML induction and consolidation?

induction: achieve remission; intensive chemotherapy for suitable patients, or approaches such as venetoclax + a hypomethylating agent for patients unsuitable for intensive treatment. Consolidation: eliminate residual disease using chemotherapy and/or allogeneic transplantation.

how do genetic risk and MRD influence AML transplantation decisions?

Adverse genetics or persistent measurable residual disease (MRD) increase relapse risk and may favour allogeneic SCT. Decisions also depend on treatment response, fitness and transplant risk. Remission does not necessarily mean eradication.

How do classical Hodgkin and non-Hodgkin lymphoma differ?

Classical Hodgkin: characteristic Hodgkin/Reed–Sternberg cells, usually B-cell derived. Non-Hodgkin: diverse B-, T- or NK-cell malignancies, with variable behaviour and more frequent extranodal involvement.

What are typical lymphoma symptoms, and how is diagnosis confirmed?

lymphadenopathy and possible B symptoms: fever, drenching night sweats and unexplained weight loss. Diagnosis requires tissue biopsy; histology and immunophenotyping establish the subtype. CT/PET helps determine extent.

What are the four Ann Arbor lymphoma stages?

I: one lymph-node region. II: multiple regions on the same side of the diaphragm. III: regions on both sides. IV: diffuse/disseminated extranodal involvement, such as bone marrow.

How do indolent and aggressive lymphomas differ?

Indolent: slow-growing, often managed as chronic disease. Aggressive: rapidly growing, requiring prompt treatment, but some are potentially curable. DLBCL is aggressive; mature-cell origin does not imply indolent behaviour.

What is R-CHOP, and what does rituximab target?

Rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone/prednisolone. It combines different mechanisms. Rituximab targets CD20 on B cells and is used in appropriate B-cell lymphomas.

How do B-cell receptor signalling inhibitors work?

They block survival/proliferation pathways used by malignant B cells. Ibrutinib: BTK inhibitor. Idelalisib: PI3Kδ inhibitor. Their suitability depends on the malignancy.

How do antibody–drug conjugates differ from bispecific T-cell engagers?

ADC: antibody delivers a cytotoxic payload, e.g. brentuximab–CD30 or polatuzumab–CD79b. Bispecific: links tumour antigen to CD3 on T cells, e.g. glofitamab–CD20 or blinatumomab–CD19.

How do CD19 CAR T cells recognise malignant B cells?

Patient T cells are engineered with a receptor that binds CD19 on target cells, activating killing through intracellular signalling domains. Recognition is HLA-independent. Normal CD19⁺ B cells can also be depleted.

What is multiple myeloma, and how is it investigated?

A malignancy of clonal plasma cells. Investigations include bone-marrow analysis, monoclonal protein/free-light-chain testing and imaging for bone lesions. CD38/CD138 indicate plasma-cell differentiation; they are not exclusive to malignant cells.

What does CRAB stand for in multiple myeloma?

C: hypercalcaemia. R: renal impairment. A: anaemia. B: lytic bone lesions. These represent organ damage attributable to the plasma-cell disorder.

What is an M-protein, and how is it useful?

a monoclonal immunoglobulin or component produced by a plasma-cell clone. It can appear as a spike on protein electrophoresis and helps monitor disease and response. An M-protein alone does not establish symptomatic myeloma.

Which main drug classes are used in myeloma?

Proteasome inhibitors: bortezomib. Immunomodulatory drugs: lenalidomide. Steroids: dexamethasone. Anti-CD38: daratumumab. BCMA-directed approaches: bispecific antibodies and CAR T cells. Combinations aim for prolonged disease control.

How do autologous and allogeneic SCT differ?

Autologous: the patient’s cells restore blood production after high-dose therapy; used in selected myeloma/lymphoma patients. Allogeneic: donor cells also provide a graft-versus-leukaemia/tumour effect, but introduce risks such as GvHD.

Which abnormalities characterise the main myeloproliferative neoplasms?

PV: excessive erythrocyte production. ET: excessive platelets. Primary myelofibrosis: marrow fibrosis with abnormal haematopoiesis. CML: excessive granulocytic proliferation associated with BCR::ABL1.

What drives CML, how is it treated, and how can it progress?

t(9;22), the Philadelphia chromosome, creates BCR::ABL1, a constitutively active tyrosine kinase. TKIs such as imatinib inhibit it. Additional abnormalities can cause progression to blast phase, which may be myeloid or lymphoid.

Why perform haematopoietic stem-cell transplantation (SCT)?

To restore blood production after intensive treatment, replace dysfunctional marrow, rebuild immunity or provide donor-derived antitumour immunity.

How are peripheral blood stem cells collected?

G-CSF mobilises stem cells from marrow into blood, followed by leukapheresis. The collected product contains stem cells and other leukocytes; most remaining blood components return to the donor.

What is the main purpose of autologous SCT in lymphoma or myeloma?

The patient’s collected stem cells rescue haematopoiesis after high-dose chemotherapy. The chemotherapy provides the main antitumour effect; there is no donor-derived GvL effect.

Why is autologous SCT unsuitable for many malignant stem-cell disorders?

The patient’s stem-cell compartment may contain the malignant clone, which could be reinfused. Allogeneic SCT supplies donor haematopoiesis and an additional immune attack against residual malignancy.

What does conditioning before allogeneic SCT achieve?

Suppresses recipient immunity to reduce graft rejection, facilitates donor engraftment and may reduce malignant cells. Conditioning intensity varies; it is not always fully myeloablative.f

Does normalisation of blood counts mean full immune recovery?

No. Neutrophils and some innate populations recover relatively early, while adaptive immunity—particularly CD4⁺ T-cell recovery—can take much longer. Infection susceptibility can persist.

ow do graft rejection, GvHD and GvL differ?

Rejection: recipient immunity attacks donor cells. GvHD: donor immunity attacks healthy recipient tissues. GvL: donor immunity attacks recipient leukaemia cells—the desired effect.

Why is HLA matching important, and can partially matched donors be used?

Matching reduces harmful alloreactivity and rejection risk. Matched siblings, unrelated donors and haploidentical donors may be used with appropriate protocols. A haploidentical donor shares one HLA haplotype.

Why can GvHD occur despite a complete HLA match?

Donor and recipient can differ in minor histocompatibility antigens (MiHAs): peptides from polymorphic proteins presented by shared HLA molecules. Matching HLA does not make all presented peptides identical.

What determines whether recognition of recipient MiHAs causes GvL or GvHD?

Where the antigen is expressed. Targets restricted to recipient haematopoietic cells may enable GvL; targets also present on healthy non-haematopoietic tissues can cause GvHD.

How does conditioning promote GvHD?

Tissue damage → inflammatory signals and APC activation → donor T-cell activation → cytokine production and tissue killing. This creates an amplifying inflammatory cycle.

Which organs are commonly affected by GvHD?

Skin, gastrointestinal tract and liver are major targets. Other sites, including mouth and lungs, can be involved, particularly in chronic GvHD.

What is the main trade-off of donor T-cell depletion

It reduces GvHD, but can weaken GvL, increase relapse risk and delay immune recovery. Donor T cells contribute to both benefit and harm.

Which approaches reduce donor T-cell alloreactivity?

TCRαβ depletion, CD34⁺ selection and lymphocyte-depleting antibodies such as alemtuzumab (anti-CD52). Post-transplant cyclophosphamide controls alloreactive responses after infusion; it does not simply remove every T cell.

What is donor lymphocyte infusion (DLI)?

infusion of additional lymphocytes, especially T cells, from the original stem-cell donor to strengthen GvL. It primarily supplies immune cells, rather than a new stem-cell graft. Its main risk is GvHD.

Why delay DLI after transplantation?

Waiting allows conditioning-related tissue damage and inflammation to subside, reducing activation against healthy tissues. Recipient APCs also become progressively replaced by donor APCs. GvHD risk remains possible.

What is the timing dilemma with DLI?

Earlier DLI: earlier antileukaemic protection, but greater GvHD risk. Later DLI: more tissue recovery, but the malignancy may relapse before immunity is strengthened.

What are prophylactic, pre-emptive and therapeutic DLI?

Prophylactic: prevent relapse in high-risk patients without detectable disease. Pre-emptive: respond to warning signs such as MRD or concerning chimerism. Therapeutic: treat overt relapse, often alongside disease-reducing treatment.

What is mixed chimerism, and does it automatically mean relapse?

Both donor- and recipient-derived haematopoietic cells remain. It does not automatically indicate relapse; significance depends on the cell lineage, trend and disease context.

How might GvL be strengthened while limiting GvHD?

Combine appropriate timing with T cells targeting haematopoietic-restricted recipient antigens. Outcome depends on antigen presentation, donor T-cell specificity, antigen distribution and inflammation. The aim is selective antileukaemic immunity.

Which classical HLA molecules present peptides to CD8⁺ and CD4⁺ T cells?

HLA-I: A, B and C → CD8⁺ T cells, mainly presenting intracellular peptides. HLA-II: DR, DQ and DP → CD4⁺ T cells, mainly presenting peptides from proteins processed in vesicles.

Why do different HLA alleles present different peptides?

HLA is highly polymorphic, especially in its peptide-binding groove. Different alleles have different binding preferences and therefore present different peptide repertoires.

What is a minor histocompatibility antigen (MiHA)?

A polymorphic peptide that differs between donor and recipient and can be recognised by donor T cells when presented on recipient HLA. HLA can be matched while the peptide differs.

How does recognition of mismatched HLA differ from MiHA recognition?

HLA mismatch: donor T cells recognise a foreign peptide–HLA complex involving a different HLA molecule. MiHA mismatch: donor T cells recognise a different peptide presented by shared HLA.

What does a 10/10 HLA match usually include?

Matching both alleles at HLA-A, -B, -C, -DRB1 and -DQB1. It does not automatically include HLA-DPB1, so a 10/10 donor may still be DP-mismatched.

Which donor T cells can respond after an HLA-matched transplant?

CD8⁺ cells: MiHAs on shared HLA-I. CD4⁺ cells: MiHAs on shared HLA-II. If HLA-DP is mismatched, additional CD4⁺ alloreactivity may occur.

Which direction of MiHA mismatch enables donor T cells to attack recipient cells?

The relevant antigen must be present in the recipient and absent from the donor. A genetic difference in the opposite direction does not provide that target for donor-derived immunity.

If only the T allele generates a MiHA, which recipient–donor genotypes provide the relevant mismatch?

recipient TT or TC; donor CC. Recipient CC with donor TT does not qualify: the recipient lacks the T-derived target peptide.

How do you calculate a directional MiHA mismatch frequency?

For randomly paired individuals, if antigen-positive frequency is p, then recipient positive × donor negative = p(1−p). Example: 0.25 × 0.75 = 18.75%. Shared restricting HLA is also required.

What are H-Y antigens, and which donor–recipient combination can generate responses?

MiHAs derived from Y-chromosome-encoded proteins. Female donor → male recipient can generate donor T-cell responses against H-Y peptides. The reverse pairing does not create the same H-Y-directed response.

Why do only a small fraction of genetic differences generate recognised MiHAs?

The source gene must be expressed, a suitable peptide must be generated and presented by HLA, and donor T cells must have a TCR capable of recognising that complex. A sequence difference alone is insufficient.

How does MiHA tissue distribution determine GvL versus GvHD?

Broadly expressed MiHAs: may cause both GvL and healthy-tissue damage. Haematopoietic-restricted MiHAs: can promote GvL with less GvHD because non-haematopoietic organs lack the target.

Why can haematopoietic-restricted MiHA targeting spare the new blood system?

After successful donor engraftment, normal haematopoiesis is donor-derived and lacks the recipient-specific target. Residual recipient leukaemia cells may retain it and remain susceptible.

What initiates donor T-cell responses against MiHAs after transplantation?

Recipient professional APCs, activated in the inflammatory transplant environment, can present recipient MiHAs and prime donor T cells. These cells can then attack tissues presenting the same targets.

What distinguished MiHA responses in patients with versus without GvHD in the presented research?

gvHD was associated with higher MiHA-reactive T-cell frequencies, particularly against targets expressed outside haematopoietic tissues. Responses against haematopoietic targets occurred in both groups.

How could MiHA knowledge improve allogeneic SCT outcomes?

Identify relevant donor–recipient mismatches, monitor specific T-cell responses and develop T cells targeting haematopoietic-restricted MiHAs. The goal is stronger GvL with less GvHD.

Why can CAR-T work in chemotherapy-refractory lymphoma?

it uses a different killing mechanism: engineered T cells recognise a surface antigen and kill the target. Chemotherapy resistance does not automatically imply resistance to immune-mediated killing.

How does CAR recognition differ from conventional TCR recognition?

CAR: directly binds a surface antigen, independently of HLA. TCR: recognises a peptide–HLA complex and can therefore detect peptides from intracellular proteins.

What are the main CAR components and their functions?

scFv: antibody-derived antigen binding.
Hinge/transmembrane region: positioning and anchoring.
CD3ζ: activation signal.
Costimulatory domain: supports expansion, survival and persistence.

How do first-, second- and third-generation CARs differ?

First: CD3ζ alone. Second: CD3ζ + one costimulatory domain, usually CD28 or 4-1BB. Third: CD3ζ + two costimulatory domains.

Why can anti-CD19 CAR-T cause B-cell depletion?

CD19 is expressed on normal and malignant B cells. Killing healthy CD19⁺ B cells is on-target, off-tumour toxicity, potentially causing hypogammaglobulinaemia and increased infection risk.

What are the main steps in autologous CAR-T treatment?

Screening → leukapheresis → genetic modification and expansion → quality control → lymphodepletion → infusion → monitoring. Manufacturing takes time, during which disease may progress.

How do bridging therapy and lymphodepletion differ?

Bridging: controls cancer during manufacturing. Lymphodepletion: given immediately before infusion to create favourable conditions for CAR T-cell expansion and activity.

Why do some referred patients never receive CAR-T?

rapid progression, poor performance status, manufacturing/access problems or patient preference. Outcomes among infused patients therefore do not capture the entire experience of all referred patients.

Does complete remission after CAR-T mean cure?

No. It means no disease is detectable using the assessment methods. Durable remissions and survival-curve plateaus suggest long-term benefit in a subset, but follow-up is required.

What causes cytokine release syndrome (CRS)?

CAR T-cell activation triggers cytokine release, amplified by monocytes/macrophages. Endothelial activation, vasodilation and vascular leakage cause systemic inflammation. Important mediators include IL-6 and IL-1.

What are the main CRS symptoms, and how is severity graded?

Fever, hypotension and hypoxia. Grade 1: fever alone. Grade 2: low-flow oxygen and/or hypotension without vasopressors. Grade 3: one vasopressor and/or high-flow oxygen. Grade 4: multiple vasopressors and/or positive-pressure ventilation. Use the most severe feature.

How is CRS treated, and what does tocilizumab target?

Supportive care and monitoring; tocilizumab blocks the IL-6 receptor. Corticosteroids and intensive organ support are added when indicated by severity and protocol.

What is ICANS, and how does it typically present?

Immune effector cell-associated neurotoxicity syndrome: confusion, impaired attention, aphasia, writing difficulties, reduced consciousness or seizures. Severe cases can cause cerebral oedema. It often develops after CRS begins.

What mechanism contributes to ICANS?

Inflammation activates endothelium and disrupts the blood–brain barrier, allowing inflammatory effects within the CNS. Activation of resident immune cells, including microglia, contributes.

What does the ICE score assess?

Orientation, naming, following commands, writing and attention, with a maximum of 10 points. A falling score suggests neurotoxicity, but ICANS grading also considers consciousness, seizures and other neurological findings.

What is the key treatment distinction between CRS and ICANS?

CRS: think tocilizumab. ICANS: corticosteroids are central when treatment is indicated. Tocilizumab treats accompanying CRS, rather than isolated ICANS, because CNS penetration is poor.

Should toxicity treatment be withheld to preserve CAR-T efficacy?

No. Necessary early treatment should not be delayed. Measured corticosteroid/tocilizumab use can control toxicity without an apparent major loss of efficacy; effects depend on dose, timing and duration.

What are the major mechanisms of CAR-T failure?

Target loss/low expression, poor expansion or persistence, immune suppression, inadequate tumour access and resistance to killing. Loss of peptide–HLA presentation alone does not prevent conventional CAR recognition.

How might newer CAR designs improve treatment?

Dual-target CARs: reduce antigen-loss escape. Armoured CARs: add functions such as cytokine support. Programmable platforms: improve control and specificity. Allogeneic products or in-vivo engineering: aim to improve access and speed.

What are the main opportunities and challenges beyond blood cancers?

Solid tumours: heterogeneous targets, poor infiltration and suppression limit efficacy. Autoimmune disease: removing pathogenic B cells may enable an immune reset, but toxicity, infection risk and long-term durability remain important.

How do transfection and transduction differ?

Transfection: non-viral delivery of DNA/RNA, using methods such as liposomes or electroporation. Transduction: delivery using a viral vector.

Why use viral vectors for gene delivery

They can efficiently deliver genetic material into cells. Depending on the vector, expression may be transient or stable. Risks include immune responses and, for integrating vectors, insertional mutagenesis.

How do adenoviral, AAV and retroviral vectors differ?

Adenovirus: generally non-integrating, often transient and immunogenic. Recombinant AAV: mainly remains episomal; can support prolonged expression. Retroviral/lentiviral: integrate into host DNA, enabling stable expression. Recombinant AAV does not normally integrate at a specific site.

How do conventional gammaretroviral and lentiviral vectors differ in target cells?

Gammaretroviral vectors: generally require dividing cells. Lentiviral vectors: can also transduce non-dividing cells. Lentiviruses are a subgroup of retroviruses.

What do reverse transcriptase and integrase do?

Reverse transcriptase: converts viral RNA into DNA. Integrase: inserts the resulting viral DNA into the host genome.

What do the retroviral genes gag, pol and env encode?

gag: structural proteins. pol: enzymes, including reverse transcriptase and integrase. env: envelope proteins involved in cell entry.

What are the functions of LTRs and the Ψ packaging signal?

LTRs: support processes including reverse transcription, integration and gene regulation. Ψ (psi): enables vector RNA to be packaged into viral particles.

How are replication-incompetent retroviral vectors produced?

The transferred genome carries the therapeutic gene and essential vector sequences. Packaging cells supply Gag, Pol and Env separately. Target cells receive the gene but lack the machinery to produce new vector particles.

What is insertional mutagenesis?

Integrated vector DNA disrupts a gene or alters nearby gene regulation, potentially activating growth-promoting genes. Stable integration provides persistence but introduces genomic risk.

How does CRISPR–Cas9 target a gene?

A guide RNA directs Cas9 to a matching DNA sequence near a suitable PAM. Cas9 cuts DNA, and cellular repair determines the resulting edit.

How do NHEJ and HDR differ?

NHEJ: rejoins DNA without a donor template; insertions/deletions can disrupt a gene → knock-out. HDR: uses a supplied template for a precise change or insertion → knock-in.

Why remove the endogenous TCR when introducing a new TCR?

Original and introduced α/β chains can mispair, creating receptors with unintended specificity. Removing endogenous chains favours expression of the intended tumour-specific TCR.

What do TRAC and TRBC editing accomplish?

TRAC disruption: removes endogenous TCR α-chain expression. TRBC disruption: removes β-chain expression. A new tumour-specific TCR can be inserted into TRAC, potentially retaining endogenous regulation.

What does peptide–HLA tetramer binding demonstrate?

it identifies T cells whose receptors bind the intended peptide–HLA complex. It supports receptor specificity but does not by itself demonstrate tumour-cell killing.

Why does 90% modified cells after enrichment not mean 90% editing efficiency?

Enrichment selects successfully modified cells, increasing their proportion. The initial editing efficiency may have been much lower—for example, approximately 5% before enrichment in the presented experiment.

What are the advantages and limitations of targeted non-viral editing?

Advantages: targeted insertion, multiplex editing, endogenous regulation and avoidance of viral-vector production. Limitations: low knock-in efficiency, off-target changes, cost and possible need for enrichment.

What is the main difference between CAR-T and TCR-T recognition?

CAR-T: directly recognises a surface antigen, independently of HLA. TCR-T: recognises a peptide presented by a specific HLA molecule, allowing targeting of proteins inside the cell.

Which targets illustrate CAR-T use in B-cell malignancies and myeloma?

CD19: B-cell leukaemias/lymphomas. BCMA: multiple myeloma. Both can also occur on healthy cells of the corresponding lineage.

What are important adverse effects of CD19 CAR-T?

CRS, neurotoxicity and normal B-cell depletion. B-cell aplasia may cause antibody deficiency, sometimes requiring immunoglobulin replacement.

What determines the specificity introduced by TCR gene therapy?

The transferred TCR α/β pair determines which peptide–HLA complex is recognised. Effective treatment requires both the target peptide and the appropriate HLA allele.

What is on-target, off-tumour toxicity?

T cells recognise the intended target on healthy tissue. Example: MART-1/gp100-specific TCRs can damage normal melanocytes, causing skin, eye or ear toxicity.

What is off-target cross-reactivity?

The introduced receptor recognises an unintended peptide–HLA complex. Example: an affinity-enhanced MAGE-A3 TCR cross-reacted with titin, causing cardiac toxicity.

Why must related proteins also be considered during safety testing?

A receptor may recognise similar peptides from related proteins. MAGE-A3-directed therapy recognised MAGE-A12 expressed in the brain, causing neurological toxicity.

What must be shown when selecting a safe and potent TCR?

Recognition of the intended peptide after natural processing, killing of relevant tumour cells, limited healthy-tissue recognition, and retained function after transfer into other T cells.