lecture 5

Question 1

What are the most common B/T-cell related cancers?

Answer 1

• non hodgkin lymphoma (2620m/2070f cases in 2012)
• myeloma (C90) (895m/650f)
• chronic lymphocytic leukaemia (C91.1) (765 men)

Question 2

What is a lymphoma?

Answer 2

• a malignancy of lymphocytes
• presents as a solid tumour of lymphoid cells, e.g. enlargement of lymph nodes
• two basic categories of lymphoma:
• • Hodgkin’s lymphoma: diagnosed by the presence off a type of cell called the Reed-Sternberg cell
• • Non-Hodgkin’s lymphomas: a large, diverse group of B cell or T cell lymphomas
• Both have indolent (slow-growing) and aggressive subtypes, that behave and respond differently
• some B cell types include: Burkitt lymphoma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma

Question 3

What is the greatest risk factor for non-hodgkin lymphoma?

Answer 3

• age is the greatest risk factor, as most cases arise in people 60+ years of age
• very rare in people younger than 50

Question 4

What is leukaemia?

Answer 4

• cancer that starts in blood-forming tissue such as bone marrow and causes large numbers of malignant cells to be produced and enter the bloodstream
• B cel, T cell or myeloid cell leukaemias: prevalance, ~8-14/100,000 (depending on race)
• B cell types include:
• • Acute lymphocytic leukaemia (ALL): a childhood peak incidence at 2-5 years and another in old age
• • Chronic lymphocytic leukaemia (CLL): the most common type of lymphoid leukaemia is B cell-CLL, mostly adults, >50 years old, males

Question 5

What is myeloma/multiple myeloma?

Answer 5

• cancer of plasma cells ( = antibody secreting cells)
• prevalence ~4-10/100,000 (depending on race)
• often preceded by an asymptomatic, premalignant stage of clonal plasma cell proliferation: “monoclonal gammopathy of undetermined significance” (MGUS)
• MGUS is present in more than 3% of the population >50 years of age, and progresses to myelome at ~1%/year

Question 6

What has the classification of B cells historically been based on?

Answer 6

Physical observations:

• patient’s condition
• histology of tumour or involved tissues (including blood)
• karyotype of tumour cells - chromosome number and structure (are there translocations?)
• abormal accumulation of clones of cells
• more recently, flow cytometry using mAbs that recognise surface molecules (CDs) on malignant cells
• even more recently diagnosing tumours by sequencing them

Question 7

What are Reed-Sternberg cells?

Answer 7

mysterious but diagnostic giant cells found in biopsies from individuals with Hodgkin’s lymphoma, with markers of both myeloid or lymphoid cells

Question 8

What is the common pathogenesis of B cell cancers?

Answer 8

• malignant cells crowd out normal cells in limited “niches”, such as the bone marrow
• this severely limits the function of normal haematopoietic cells such as RBC and WBC that are essential to life, thereby causing infection, anaemia, platelet deficiencies, bleeding problems, and respiratory failure

Question 9

What is a diagnostic feature of myeloma patients?

Answer 9

• normal serum Ig concentration is ~10-15mg/ml
• myeloma patients have clonal “paraprotein”, in serum and urine, often»_space; 30 mg/ml, with free light chains “Bence Jones proteins” (Ig lambda and kappa)

Question 10

How do myeloma cells destroy bone marrow?

Answer 10

Normal plasma cells like to go back and sit in the bone marrow and produce antibody in a benign way for decades.
Myeloma bind to stromal cells in the bone marrow –> an imbalance between factors that control bone homeostasis:
- growth factor RANK-L (bone resorbing) is overproduced
- growth factor OGP (bone-forming) is diminished (osteogenic growth peptide)
- results in osteoclastogenesis and osteolysis

Question 11

What are the diagnostic criteria for multiple myeloma?

Answer 11

• clonal plasma cells >10% on bone marrow biopsy
• a monoclonal antibody in either serum or urine
• end organ damage:
• • bone lesions and fractures
• • hypercalcemia
• • renal insufficiency
• • anaemia
• • frequent severe infection

Question 12

What are the current therapies for patients with Non-Hodgkin lymphoma?

Answer 12

1. chemotherapy (systemic): general toxins that kill rapidly dividing cells - CHOP
   - standard care for decades
2. Antibody - Rituxan - R-chop
   - monoclonal antibody
   - made in a lab
   - injected into patients
   - specific for B cells
   - targets both cancerous and normal B cells for destruction by the patients own IS
3. Radiation - limited
4. Bone marrow transplantation - if things are really dire, all blood cells killed off and replaced by normal, healthy donor stem cells to give them effectively a new IS, which will provide protection against infection, and in many cases will also fight the cancer cell, which it will see as foreign

Question 13

What is CHOP?

Answer 13

- the chemotherapy regimen used in the treatment of non-Hodgkin lymphoma. Consists of: 
Cyclophosphamide
Hydroxydaunorubicin
Oncovin
Prednisone

Question 14

What is an example of targeted therapies to suit multiple myeloma?

Answer 14

First choice therapies include thalidomide and lenalidomide (a derivative)

• thalidomide reasonably effective: targets the energy production of cells
• plasma cells are sensitive to this: they need a lot of energy, are producing a lot of proteins
• other treatments include melphalan, corticosteroids, autologous transplantation, bortezimib
• none of these are curative - there is no cure, simply managing the disease so that symptoms don’t get too severe

Question 15

What is Bortezimib?

Answer 15

• a drug used in the treatment of MM
• a boronic acid dipeptide
• Inhibits the 26S proteosome - a large protease complex that collaborates with the ubiquitin system to mark proteins for degradation including key regulatory proteins and transcription factors including NFkB (also their activation relies on proteasomal degradation of some component of them)
• Plasma cells (and myeloma cells) are protein secreting factories - handling large quantities of immunoglobulin - so are especially sensitive to this drug
• NFkB is required for expression of the survival genes on which MM rely
• no better molecular therapies - yet
• proteasomes are present in every cell so it is important to try and get the dosage right, so that it is mostly the myeloma cells that are effected
• has a lot of side effects

Question 16

What is another therapy used for MM that slows growth and relieve symptoms of fragile bones?

Answer 16

• blocking bone destruction with bisphosphonates - slow MM growth

Question 17

What is a treatment specific for B cell leukaemia and lymphoma?

Answer 17

• Rituximad - a monoclonal anti-CD20 antibody
• CD20 - a surface protein with no known ligand. Function unclear - may act as a Ca+ channel
• All mature B cells express CD20 (not plasma cells)
• Rituximab depletes all CD20+ B cells (transiently)
• First trials (‘97/’98) on “non-responding” patients: ~50% partial or complete remission
• non-toxic, highly efficacious, “blockbuster” drug
• If a normal adult is given three doses separated by two weeks they will not have B cells in the periphery for up to a year
• very useful as an adjunct to cancer therapy
• not a cure

Question 18

What are some of the features of DLBCL?

Answer 18

• post GC B cells - switched and mutated Ig
• Clinically heterogeneous: 40% of patients respond well to current therapy with prolonged survival, while the remainder succumb to the disease
• distinct types of diffuse large B-cell lymphoma identified by gene expression profiling of many tumours (the present and future of cancer diagnosis: personalised medicine)
• different pathways are hyperactive in the two types of DLBCL
• it is possible to tell which type of DLBCL a patient has through doing a microarray with a small subset (~6) of the most telling genes –> a “signature”
• GC type and ABC type

Question 19

How has distinguishing between the types of DLBCL effected diagnosis, prognoses and treatments?

Answer 19

• by traditional methods those patients in the low clinical risk survived much better than those in the high clinical risk in terms of overall survival in years from the point of diagnosis
• if you do a gene segregation study of patients you find that GC B-like survive better than Activated B-like
• if you take the original, low clinical risk group, most of them are GC-B-like but still some of the activated B-cell type
• therefore those in the GC-B-like category have extremely good survival. The benefit of knowing this is that you would only need to give these people relatively mild or conventional therapy, conversely those in the other group you would want to treat in the most aggressive way that you possibly could
• the two gene expression patterns also suggest that different drugs will be effective in these two types of
  DLBCL:
  – standard chemotherapy (+Rituximab) in low risk
  – specific enzyme inhibitors targeting hyperactive pathways in high risk

Question 20

What are some treatments for B cell malignancies based on drugs targeting survival?

Answer 20

• cell survival vs death is a tug of war
• BH3-only = pro-survival antagonist
• In a state of equilibrium, with BCL-2 pro-survival family
• Bcl-2-like proteins hold “death” signals in check but are often over-expressed in B cell cancers
• Danger or damage signals would normally remove/enhance the transcription of pro-survival antagonists from/over proteins that are blocking them
• These go on to initiate cell death cascade “apoptosis”
• The drug ABT-263 (Navitoclax) blocks the binding site of Bcl2-like proteins, therefore releasing the death inducer and triggering apoptosis

Question 21

On which groups of patients should ABT-263 work effectively?

Answer 21

• Bcl-2 overexpression is most common in Follicular and CLL lymphomas - 90% of patients with these cancers
• human trials of ABT-263 (and derivative 199) are going very well
• CLL patients respond to treatment with BH3-mimetic ABT-199:
• • 3 CLL patients given a single dose and examined 24 hours later
• • ABT-199 induced almost complete disappearance of CLL cells in blood while leaving platelets unaffect
• • ABT-263 (navitoclax) is less effective on CLL and kills platelets, leading to bleeding

Previous Navitoclax trial and RMH:

• lymphocytosis was reduced by more than 50% in 19 of 21 patients with baseline lymphocytosis
• median treatment duration was 7 months (range, 1 to >= 29 months)
• median progression-free survival was 25 months
• you would think that triggering systemic apoptosis would be pretty bad but apparently through Bcl-2 pathway it is not that toxic: people can be on these compounds for quite considerable times

Question 22

What are the present and future of cancer diagnosis?

Answer 22

• better diagnosis, treatment and prognosis based on molecular profiles of each patient’s tumour
• identification of cellular processes that are deregulated in individual cancers
• rationally designed, SPECIFIC drugs that address the gene mutation driving the cancer, and spare normal cell function

Question 23

What are monoclonal antibodies? Why do they have great therapeutic potential?

Answer 23

• mAbs are a pure population of antibodies with one target specificity made by a clonal population of antibody secreting plasma cells
• great therapeutic potential:
• • extremely well-tolerated: serum is >10mg/ml immunoglobulin
• • long-lived: normally a half-like of several weeks
• • very specific and selective: binding constants >= 1/10^9 M for their targets

Question 24

What are some of the therapeutic applications of mAbs as approved by the US food and drug administration?

Answer 24

• malignancies
• autoimmune conditions
  (the first two make up ~80% of the current market)
• transplant rejection (anti-T cell)
• infectious disease (respiratory syncytial virus)
• cardiovascular disease (glycoprotein IIb/IIIa)
• macular degeneration (VEGF-A)
• paroxysmal nocturnal hemoglobinuria (complement C5)

Question 25

What are the biggest monoclonal antibodies in use?

Answer 25

- Anti-TNF: infliximab (remicade), adalimumab (Humira) used to treat Rheumatoid arthritis, Crohn's disease, psoriasis - Anti-HER2: trastuzumab (herceptin) used to treat cancer, especially breast - Anti-VEGFA: bevacizumab (Avastin) used for anti-angiogenesis, cancer therapy - Anti-CD20: rituximab (Rituxan) used to treat Rheumatoid arthritis and non-Hodgkin's lymphoma these 5 earned > US$37 billion in 2012 also Enbrel (etanercept), soluble form of the TNF receptor for RA

Question 26

Where were mAbs first created?

Answer 26

- lab in Cambridge by Köhler and Milstein - won the nobel prize for medicine in 1984 - fused mouse myeloma cells with mouse spleen cells from an immunised donor giving a B cell, specific for a particular antigen, immortality

Question 27

What was the original strategy for making mAbs?

Answer 27

- take myeloma cells that have lost the ability to produce their own antibody and force them to fuse (including the nuclei) with the spleen - you select from the hybridomas those cells that have the Ig producing genes and the ability to grow forever from the myeloma cells - occasionally the one that you are interested in will be included - recover this cell, isolate it, grow it on its own and you now have an ever growing single cell type that is making an antibody that is useful to you

Question 28

What were the short comings of the first generation of mAbs that were raised in mice?

Answer 28

- very specific and high affinity, unlimited quantities BUT - seen as foreign in humans and eventually "rejected" - short serum half-life - because their constant region domains are mouse-derived --> lack some important effector functions in human

Question 29

Why do we 'humanise' mAbs?

Answer 29

- to avoid anti-mAb immune reaction in the patient