LV Flashcards

Question

Allogeneic HSCT rationale? What is the importance of alloreactive T cells?

Answer 1

Allogeneic HSCT works not only by replacing the diseased marrow but also through the graft-versus-leukemia (GvL) effect, driven by alloreactive T cells that recognize and attack residual tumor cells. This immune response is possible due to genetic differences between donor and recipient—but these same differences can also cause graft-versus-host disease (GvHD). In malignant diseases, some degree of alloreactivity is beneficial for anti-tumor effects. In non-malignant diseases like aplastic anemia, genetically identical donors are preferred to avoid GvHD, as GvL is not needed.

Answer 2

HLA is a large region of the genome (4 million base pairs) that over time was constantly improved to better protect ourselves against infections. We generate a lot of HLA genes, and each of them can evolve independently. This system is, therefore, mostly characterized by polymorphism, leading to different individuals having diffrent HLAs. We have 20,000 described alleles for class 1 and at least 6,000 for class 2, not all of them are equally frequent in different individuals and some are more frequent in one population than another.

Answer 3

A haplotype is a group of alleles that are inherited together from a single parent because they are located close to each other on the same chromosome (in cis). Since HLA genes are tightly clustered on chromosome 6, they are usually inherited as a block—one haplotype from each parent. This is important in transplantation because matching entire HLA haplotypes increases compatibility. Due to this inheritance pattern, siblings have: • 25% chance of being fully HLA identical, • 50% chance of being haploidentical (sharing one haplotype), • 25% chance of being completely mismatched.

Answer 4

100%. By definition, parents and children are haploidentical, since both parents transfer a haplotype.

Answer 5

Considering that we have 6 main HLA genes: HLA A, B, C, DRB1, DPB1, and DQB1; one of them (DP) is not considered as criteria for matching, because its relative weight on clinical outcome is much lower. HLA DP is farther away from all the other genes on the chromosome, it is more likely to be different, so people who are matched in the other 5 genes may not be compatible with DP. Unrelated donors can be: • Matched (10/12 loci) • Mismatched (8/12 loci) • Umbilical cord donors (being immunological naive, it allows for more incompatibility) When talking about matching we have to take into account that even if we have a lot of polymorphisms in the HLA, not all differences will have the same contribution to the outcome of the patient. In general, the more mismatches we have the more likely that the graft would cause a reaction. There have been cases where a difference in one amino acid was enough to either cause a reaction or not. Even the position of that AA is important.

Answer 6

1. Conditioning Phase (1–2 weeks): High-dose chemo ± radiation to create marrow space, immunosuppress the host, and eradicate disease. Risks: toxicity, mucositis, early rejection, infections. 2. Stem Cell Infusion: IV infusion of donor HSCs that home to bone marrow. Risks: infusion reactions, graft rejection, severe neutropenia → infections. 3. Immune Reconstitution Phase (months–years): Engraftment followed by slow immune recovery. Risks: GvHD, infections, disease relapse.

Answer 7

Patients with a competent immune system, such as those with thalassemia or sickle cell disease, have a higher risk of transplant rejection due to factors like prior transfusions, intact immune responses, and bone marrow fibrosis requiring intensive conditioning. In contrast, immunodeficient patients—particularly those with defects in T-cell development, like SCID—are less likely to reject a transplant because they cannot mount an effective immune response. Similarly, patients with aplastic anemia who haven’t had many transfusions also have a lower risk due to reduced immune activity and minimal alloimmunization.

Answer 8

Direct action of the chemo radiotherapy : Nausea, vomiting, diarrhea. Specifically cyclophosphamide can cause hemorragic cystitis, lung toxicities can occur although rare. Vascular endothelial syndrome can also occur VOD, thrombotic microangiopathy, engraftment syndrome and capillary leak syndrome. Other complications include : Drug toxicities, infections and immune complications.

Answer 9

Hepatic veno-occlusive disease, also known as sinusoidal obstruction syndrome (SOS), is a well-recognized and potentially life-threatening complication that occurs primarily after myeloablative hematopoietic stem cell transplantation. VOD/SOS arises from endothelial cell damage and hepatocellular injury due to the transplantation conditioning regimen. The complex pathogenesis begins with injury to sinusoidal endothelial cells and hepatocytes due to toxic metabolites generated by high-dose alkylating chemotherapy conditioning regimens, such as busulfan, cyclophosphamide and melphalan. VOD/SOS is clinically characterized by fluid retention and ascites, jaundice, weight gain (≥5%), and painful hepatomegaly, in the absence of other identifiable causes of liver disease. In VOD/SOS hepatomegaly occurs quickly accompanied by right upper quadrant pain while in GvHD there is no pain and hepatomegaly. Other DDX may be CHF, kidney failure, infections.

Answer 10

Low molecular weigh heparin was once recommend as prophylaxis for VOD/SOS, now it is not recommended because of the significant risk of hemorrhages. The only approved drug for treatment in Europe is Defibrotide. It is a mixture of oligonucleotides derived from porcine intestinal mucosa. It has been described to have an anti-inflammatory action on the endothelium. It’s main risk is hemorrhages.

Answer 11

Regeneration of a functional immune system requires recovery of innate and adaptive components and occurs with different kinetics. While most branches of the innate immune system are restored rapidly, the recovery of a broad functional T and B lymphocyte repertoire is much more difficult to achieve. Recovery in cell counts does not necessarily correlate with recovery in cellular function. The thymus is important in building our immune system but, since most of the patients are 60 -70 years old, they don’t have a functional thymus so we will have to rely on what was transferred.

Answer 12

This is an immunological disease that is uniquely related to transplantation and affects many organ systems, the main ones being: the GI tract, the liver, the skin, and the lungs. Pathophysiology : The transplanted T cell recognize the hosts APCs as an infection and launch an immune response. The T cells then, in a secondary lymphoid organ, expand and differentiate to increase to strength of the immune response. This leads to increased inflammation and death. Many different cytokines and effectors are involved. Clinical manifestations : In the acute phase of GvHD 3 organs are affected: the skin, the GI tract, and the liver. Manifestations include maculopapular skin rashes in the skin, nausea, vomiting and diarrhea for the GI tract, cholestatic hyperbilirubinaemia and VOD for the liver.

Answer 13

Acute GvHD can turn into chronic GvHD or it can arise De Novo, but the pathophysiology is a bit different : whereas in acute GvHD the initial priming comes from inflammation, in chronic GvHD it’s more a reaction in which a moderate recognition of some antigens leads to chronic inflammation and chronic activation of the immune system. There might be subtle manifestations, like fibrosis in the skin, dry skin, lichen type lesions, etc. The more the differences between the donor and the patient the higher the risk of GvHD. The main contributor is therefore not proper HLA matching.

Answer 14

The number of T cells in the graft plays a critical role in the outcome of HSCT. A high T cell dose increases the risk of GvHD while a low T cell dose is associated with higher rates of infection and disease relapse, due to insufficient immune reconstitution and weaker graft-versus-leukemia. There are different strategies to modulate the T cells numbers : 1. Anti-thymocyte globulin (ATG): Administered pre-transplant to deplete host and donor T cells, thereby reducing the incidence and severity of GvHD. It is relatively specific in its action. 2. Suicide gene-modified T cells: Donor T cells can be genetically engineered with a “suicide gene,” such as HSV-thymidine kinase (HSV-TK). These cells are expanded in vitro and infused post-transplant (typically after 20–40 days) to support immune recovery and GvL. If GvHD occurs, an antiviral drug like acyclovir can selectively eliminate the engineered T cells. However, HSV-TK use has declined due to concerns about antiviral resistance and toxicity. iCaspase9, an inducible suicide switch activated by a small molecule drug, is now being explored as a safer alternative. 3. T cell subset selection: T cells differ by receptor type—alpha-beta T cells are the main mediators of GVHD, while gamma-delta T cells and NK cells may contribute to anti-infective and anti-leukemia effects without causing GVHD. Modern graft engineering involves depleting alpha-beta T cells and B cells, while preserving or enriching gamma-delta T cells and NK cells to enhance immune protection and reduce GVHD risk.

Answer 15

Rapamycin inhibits the mTOR (mechanistic target of rapamycin) pathway, which is crucial for cell growth, proliferation, and survival. By blocking mTOR, rapamycin suppresses T cell activation and proliferation, making it useful in preventing organ transplant rejection and in graft-versus-host disease (GVHD) prevention in HSCT. Uses : immunosuppression, GvHD prophylaxis, cancer therapy and longevity research.

Answer 16

Increased compatibility although decreases the risk of GvHD it increases the chance of the disease relapsing. In a mouse model it was demonstrated that leukemia was better eradicated in haploidentical or mismatched rather than HLA identical.

Answer 17

In HSCT, leukemia relapse often occurs through a mechanism called copy-neutral loss of heterozygosity (CN-LOH). This genetic alteration allows leukemic cells to lose the mismatched HLA haplotype inherited from the donor and replace it with a duplicated version of the patient’s matched HLA. As a result, the leukemia cells become HLA-compatible with the host, making them invisible to donor-derived immune cells—a form of immune escape. Although relapses rates are generally less common haploidentical HLA, this mechanism is the most common among haploidentical transplants due to the strong HLA mismatch pressure. In contrast, relapse rates are lower with matched sibling or unrelated donors, where fewer mismatches exist and the immune system can recognize leukemia through additional non-HLA antigens, making immune escape less likely via this pathway.

Answer 18

- Solid tumors present with high number of mutations. This leads to a lot of branching, complexity but also vulnerability by generating a lot of antigens and becoming more susceptible to the immune system. -Hematological tumors have low number of mutations. This is because they don’t have to acquire all the mutations needed for angiogenesis, tissue invasion, contact inhibition. They are considered systemic pathology. The consequences of having few mutations are : Homogeneity of the tumors (low branching, low complexity), all the mutations are fundamental for the biology of the tumor. The fewer the mutations, the easier is the eradication of the tumor.

Answer 19

They are mutations that are the founders of the disease. All patient with these diseases carry the mutation, all tumor cells carry the mutation and blocking the effect of the mutation can cure the disease. Some examples are BCR/ABL translocation which causes CML. Inhibitors of the tyrosine kinase of the ABL gene is used to treat the disease. Another example is the PML/RAR alpha mutation in acute promyelocytic leukemia, a subtype of AML. These specific mutations for these specific diseases are both founders and drivers of the disease itself.

Answer 20

A gate keeper mutation makes the difference between a cell being normal and a tumoral cell. An accelerating or driver mutation is responsible for certain important aspect of the disease, but inhibiting is not enough to cure the disease. An example is the BCR/ABL mutation in ALL or the FLT3 mutation in AML.

Answer 21

Clonal myeloid disorders arise from pathological transformations in early hematopoietic progenitor cells, typically at the level of hematopoietic stem cells (HSCs) or common myeloid progenitors (CMPs). These mutations lead to disorders such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs). Lymphoid malignancies, on the other hand, can originate at multiple stages of lymphocyte development, as lymphoid cells undergo extensive genetic rearrangements and maturation: • Mutations in early lymphoid progenitors (in the bone marrow) can lead to acute lymphoblastic leukemia (ALL). • Abnormalities in maturing B or T cells, especially those that migrate to secondary lymphoid organs (like lymph nodes), can result in lymphomas or chronic lymphocytic leukemia (CLL). • Plasma cells, which are terminally differentiated B cells that return to the bone marrow, can undergo malignant transformation, giving rise to multiple myeloma.

Answer 22

With clonal myeloid disorder we refer to acute myeloid leukemia, myelodispastic syndrome and myeloproliferative neoplasia. These diseases they originate from a same founder mutation/first hit depending on the type of gate keeper event/second hit, we can have the progression of the three disorders. The common founder event is DNMT3A, TT2 or IDH1/2 mutation. The progression depends on the gate keeper event : NPM1 or FLT3 mutations gives rise to AML, spliceosome complex genes mutations give rise to MDS and JAK2 mutations give rise to MPN. An important information is that if MDS or MPN patients accumulate additional damage they can evolve in secondary AML.

Answer 23

AML is a clonal malignancy of hematopoietic progenitor cells from the myeloid lineage, or from cells that acquire similar characteristics. It is characterized by the accumulation of immature myeloid blasts in the bone marrow, leading to uncontrolled proliferation and impaired hematopoiesis. These blasts infiltrate the marrow, causing secondary bone marrow failure. Epidemiology and prognosis : AML is a highly aggressive leukemia with a significant mortality rate — approximately 50% overall. Prognosis is especially poor in elderly patients; the median age at diagnosis is ~70 years, and many patients are over 80, making them less eligible for intensive therapy. Clinical presentation : neutropenia, thrombocytopenia, anemia, constitutional symptoms like fatigue, tachycardia and malaise, leukemic infiltration causing soft tissue masses. Diagnostic work up based on CBC, circulating blasts are always pathologic so peripheral blood smear is essential, coagulation tests, BM biopsy.

Answer 24

Leukemic blasts are large-sized cells with a high nucleus/cytoplasm ratio. They are basically indistinguishable from HSCs. After performing chemotherapy, for example, we can’t be sure whether the bone marrow is clean again or not. The only distinguishable feature that allows us to tell with certainly that we are not looking at healthy cells is the Auer rod made of granules. They are present only in malignant cells.

Answer 25

FAB : French American British classification considers only morphology as a way to classify AML. If the cells look very immature M0 if they look very mature resembling a megakaryocyte then M7. M0 and M7 are both the two extremes of the classification and although completely different they both have very bad prognosis. WHO : They consider morphology, immunophenotypes, cytogenetic and molecular genetics. Flow cytometry and antibodies can be used to characterize the profile of cells at different stages of maturation, which can be obtained through the expression of different markers. For example : -CD34 marker stains hematopoietic stem cells. -CD14 stains mature monocytes and neutrophils. Using flow cytometry to identify the leukemia associated immunophenotype is important : - Asynchronous expression : CD34 is the classical marker of HSC, CD14 is for monocytes, if we have co-expression of the two, this is an asynchronous expression, because in physiology you’ll never have the co-expression of these two markers. If you find this phenotype in a patient, after having treated it, you can follow this phenotype and look specifically for the persistence of these cells. -Cross linkage expression : Markers from myeloid linkage and from lymphoid linkage co-exist in a cell. When cells with both markers is identified it will signify that the cell is not normal.

Answer 26

Patient related factors such as age, performance statutes (how well he or she is able to perform daily activities), and comorbidities like cardiovascular diseases or type II diabetes. Disease related factors such as cytogenetics which can be favorable, unfavorable or intermediate. Genetic mutations are important as well. DNMT3A, FLT3 and NPM1 are the most frequent. They are imprint as different mutations have different prognosis. Therapy related factors include chemoresistance, relapse and minimal residual disease ( disease persistence below level fo detection of morphological examination). All these factors have to be integrated to classify the patient : low, intermediate or high risk.

Answer 27

Induction chemotherapy—> It induced the remission of the disease, but does not mean the patient is cured. Consolidation chemotherapy—> It is a combination of only chemotherapy or chemotherapy with the addition of BM transplant. It is done to avoid relapse. Chemotherapy is mostly based on two drugs that have not changed in years : Ara-C and antracycline.

Answer 28

- DH1 and IDH2 inhibitors, which are important for the epigenetic of the disease but are infrequent (10%). - FLT3 inhibitors, very important because this mutation is present in 45% of patients. Unfortunately, this mutation is not curative, because it is not a driver mutation. We can only achieve a slow down of the disease. If we keep using only these without targeting the driver mutation, we will lead to changes in some domains of the receptor, leading to a condition of resistance. They must be used carefully and integrated with other strategies. - Bcl2 inhibitors, which activates apoptosis. Most common option for elders. - Demethylating agents (azacitidine, decitabine), they interfere with the methylation of DNA, activating the tumour suppressor genes leading in the slowing down of a not so aggressive /proliferative disease.

Answer 29

It is a subtype of AML. It accounts for 10-20% cases of AML. The first distinguishing feature is that it presents at a much younger age, at 30-40 years of age (classic AML is usually diagnosed around 70 y.o), with a single mutation that is sufficient for the transformation. Morphologically, it is characterised by promyelocytes, that are cells full of granules, making them very easy to recognize. The clinical presentation is dramatic. The granules are very rich in proteins related to the coagulation system and are able to activate the DIC (disseminated intravascular coagulopathy) process. If DIC is frequent but controllable in AML, in PML it is always invariably present at lab level and often severe at a clinical level. From the biological point of view, we recognize a translocation from chromosome 15 to chromosome 17 – t (15,17), bringing PML and RARalpha genes together. This translocation founder and driver of the disease.

Answer 30

All-trans retinoic acid (ATRA), a vitamin A derivative, is a cornerstone treatment for acute promyelocytic leukemia (APL), characterized by the PML-RARα fusion gene. This fusion protein blocks myeloid differentiation by recruiting co-repressors. ATRA binds RARα, displaces the co-repressors, and restores transcriptional activity, allowing differentiation of promyelocytes into mature neutrophils. Unlike traditional chemotherapy, ATRA does not directly kill leukemic cells — instead, it removes their selective advantage, leading to terminal differentiation and eventual apoptosis. During therapy, you can observe blasts maturing into polymorphonuclear neutrophils. Eradication of every leukemic cell isn’t necessary — simply disrupting the leukemic program can be enough for cure. The efficacy of ATRA is potentiated by arsenic trioxide (ATO), which also targets PML-RARα. The combination is highly effective but may cause cardiac toxicity, particularly QT prolongation and arrhythmias.

Answer 31

It is a disease very similar to AML, sharing many characteristics. The cells that start to become sick are progenitor cells as in AML. One of the major differences is the age of onset of the disease. AML is considered a disease of the elderly while 80% of all ALL patients are children or young adolescents with a median age of 7.

Answer 32

Two phenotypes of the disease—> B cell leukemia which represent 80% of all ALLs and T cell leukemia which are much more rare and more aggressive.

Answer 33

In ALL we use cytogenetic as first level of prognostication. There are several relevant alterations other than the famous Philadelphia translocation, t(9;22) BCR-ABL1, like t(4:11) AF4-MLL, hypodiploid, triploid and complex karyotype (more than 5 alterations in the same cell). Cytogenetics often vary between pediatric and adult B- cells-ALL patients. While pediatric mutations generally lead to a good prognosis, the adults’ ones are considered “high risk” alterations.

Answer 34

⸻ Diagnosis is established through bone marrow aspiration and examination, which reveals the presence of leukemic blasts. A threshold of ≥20% blasts in the bone marrow is typically required to confirm the diagnosis of acute leukemia. In addition to identifying the blast population, it is essential to assess cytogenetic abnormalities, molecular mutations, and immunophenotypic markers to classify the subtype of leukemia and guide prognosis and treatment. Immunophenotyping (Flow Cytometry) • Differentiates B-ALL vs T-ALL Key markers: • B-ALL: CD10, CD19, CD20, CD22, TdT • T-ALL: CD2, CD3, CD5, CD7, TdT

Answer 35

The most important prognostic factors for ALL are age, sex, ethnicity, hyperleukocytosis, immunophenotype (t-cell worse than b-cell), cytogenetic features, genetic mutations and resistance to the initial treatment.

Answer 36

While for AML there are two phases of treatment, for ALL we have the following three. - Induction chemotherapy—> using chemotherapy a to start sending the disease in complete remission (<5% of immature cells in BM). Use of anthracyclines, vinecristine, prednisone, asparaginase. - Consolidation therapy—> aims to increase hematological remission. Several cycles of chemo and other different drugs are used. - Maintenance therapy—> aims to eliminate long lived reservoirs of the disease. Use of vincristine-AraC, purine analogues and methotrexate. While more than 95% of pediatric patients achieve remission and more than 85% are alive and disease-free at 5 years from initial diagnosis, no more than 40% of adult ALL patients achieve long-term complete remission.

Answer 37

Minimal residual disease is an important concept which is taken into consideration to give the most promising clinical outcomes when combines with allo HSCT for example. After induction and consolidation therapy id the MRD evaluation is neg—> maintenance, if it is positive—> auto or allo HSCT followed by another MRD.

Answer 38

- Tyrosine Kinase Inhibitors in Philadelphia+ - Blinatumomab, a specific T cell engaging antibody with anti CD3 and anti CD19. Used in patient who do not respond well to chemotherapy before doing transplantation. - CAR T cells have been approved only for children in ALL.

Answer 39

It is a clinical disease due to progressive accumulation of mutation leading to a dysfunctional bone marrow. Classification is very complex because it takes into consideration genetic markers, percentage of blasts and presence of peculiar alterations. Incidence is about 4 cases per 100.000, quite similar to AML and slightly more frequent to B-ALL. 90% of all cases occur after 60 year of age. MDS is characterized by impaired differentiation and maturation of cells, abnormal cell features and high tendency to apoptosis.

Answer 40

Clinical presentation—> manifestations are not specific since any lineage can be affected with any combination. It might start with leukopenia giving fever due to recurrent bacterial or viral infections, anemia manifesting as fatigue and exacerbation of cardiac symptoms and thrombocytopenia leading to bleeding and bruising. - MDS can be included in all DDX in which cytopenias are present. Examination—> full blood count to look for cytopenias, lab tests to look for cytopenias causes such as iron, vitamin asset, liver function, hemolysis indexes, reticulocytes count, tumor markers and viral routine. Peripheral blood smear is also performed. Bone marrow aspirate. Most common DDX is usually aplastic anemia which is differentiated because AA is autoimmune disorders and cells are non mutated while in MDS the cells have clonal mutations.

Answer 41

Chemotherapy should be avoided since this is a clonal disease of cells that do not work properly and there is no need to treat proliferation as in AML. Chemotherapy could only worsen the general BM condition, inducing further aplasia and hindering any possible pancytopenia recovery. We have a BM that does not work anymore, hence Allogenic HSCT is potentially the only curative option. The problem is that treatment-related mortality is 20% or higher (often in the first year after transplant) and increases with age. It is important to consider all possible scenarios. The patient can remain stable with anemia for example (differently from an AML), but it can happen that the cytopenias worsen or that there can even be an evolution towards secondary AML (very bad prognosis). Supportive therapies include transfusions, iron chelation or anti infectious prophylaxis. Other agents include G-CSF, luspatercept which is an antagonist of TGF-b pathway, epigenetically active drugs.

Answer 42

Neutropenia→ <1,5x10^9/L Anemia→ <10g/dL (high MVC) Thrombocytopenia→ <100x10^9/L