Hematologic Malignancies I - Strom 03.23.15

Question 1

How are hematologic malignancies diagnosed?

Answer 1

1. Clinician recognizes possible malignancy
2. Performs/requests appropriate initial tests:: CBC, peripheral smear, imaging studies
3. Clinician obtains tissue for pathologic confirmation of diagnosis: peripheral blood, bone marrow biopsy or aspirate, lymph node biopsy
4. Pathologist makes initial assessment, and orders confirmatory tests: flow cytometry, immunohistochem, cytogenics, FISH, DNA seq analysis
5. Pathologist make the dx (or not)

Question 2

White cell count 120,000 per ul. Lab calls to tell you there are blasts. Pt was well until last week. Dx?

Answer 2

• Acute leukemia

Question 3

The white cell count is normal, platelet count is low, and peripheral smear contains a small number of “atypical” cells. A bone marrow biopsy is done, and the marrow is replaced by blasts. Dx?

Answer 3

Acute leukemia

Question 4

When should you expect a hematologic malignancy?

Answer 4

1. When bone marrow is not functioning normally, and you can’t find a simpler explanation. Examples:
   - Unexplained low cells
   - Unexplained high cell
   - Cells normally found in marrow present in peripheral blood (i.e., blasts, leukoerythroblasts, or myelophthisic features)
2. When lymphatic tissues enlarged, and no infectious etiology found (splenomegaly or LAD)

Question 5

What morphologic features suggest a leukocyte is a blast?

Answer 5

1. Large cells
2. High nuclear/cyto ratio
3. Prominent, single or multiple nucleoli
4. Immature (faint, smudgy) chromatin
5. Appearance shared by many cells on a slide

Question 6

What are Auer rods always diagnostic of?

Answer 6

Myeloid blasts

Question 7

Is immunophenotype acceptable as a sole diagnostic criterion of blasts?

Answer 7

• No. Back-up to morphologic identification, but not acceptable as sole diagnostic criterion.

Question 8

How does the workup of any hematologic disease begin?

Answer 8

With a review of the peripheral smear

Question 9

What is this?

Answer 9

Nucleated RBC

Question 10

What is this?

Answer 10

• Basophilic stippling: non-specific indicator of abnormal erythroid maturation
• Howell-Jolly body: abnormal erythroid maturation or asplenia

Question 11

What is this?

Answer 11

Giant platelet

Question 12

What is this?

Answer 12

Myelocyte

Question 13

What is this?

Answer 13

Blast

Question 14

Describe bone marrow biopsy.

Answer 14

1. Prep sterile working field (usually posterior iliac crest), and inject local anesthetic
2. Drive large needle into bone, and draw out (aspirate) half cc of thick, bloody fluid containing bony spicules
3. Additional 5-20 cc’s for special studies, as needed (hemodilute: diluted with peripheral blood)
4. Large needle at very different angle or different site for core biopsy (NOTE: if same site as aspirate, will be empty of blood-forming elements)

Question 15

What does a bone marrow aspirate report look like?

Answer 15

• Pathologist counts different cell types present for the differential count
• Blasts normally make up < 5% of cells present, and myeloid cells (granulocytes, plus monocytes) should outnumber erythroid precursors by 2:1 to 5:1
• Manual count of cell types in bone marrow aspirate is currently “gold standard” for determining if there is an abnormal proliferation of blasts or not

Erythroid cells look like bowling balls here, with very little cytoplasm. Of course, there are more myeloid cells here than erythrocytes.

Question 16

What does the pathologist look for in a bone marrow biopsy?

Answer 16

• Specimen adequacy (should be 2-3cm long)
• Cellularity estimate (< with age; should be about 100-age)
• Myeloid:erythroid ratio
• Iron stores estimate
• Any abnormal cell types (by appearance)

Question 17

What is the blue in this bone marrow core biopsy?

Answer 17

Iron compounds (these should be here)

Question 18

What is the significance of this image?

Answer 18

• Different cell types express different antigens that can be detected with monoclonal Abs specific for each antigen
• Hundreds of them are characterized, & commercially availablle. Antigenic profile of cell in most cases defines its functional nature
• Most Ags referred to by their “cluster designation” (CD). A CD entity is a hapten, a site where an Ab binds -> 1 protein can have more than one CD designation

Question 19

What is immunophenotyping?

Answer 19

• Flow cytometry to count cell types in bone marrow aspirate -> lyse red cells and add fluorescent Abs to desired cell surface proteins (most labs start by plotting CD45 - lympho common Ag - vs. SSC)
• 2 things to bear in mind:
  1. Aspirate used for this is normally hemodilute
  2. Red cell lysis procedure lyses most or all erythroid precursors (so flow cytometry a weak method for characterizing these cells)

Question 20

What is measured by flow cytometer?

Answer 20

• Several optical measurements from e/cell passing though its “flow cell”; dozens of cells per second
• Most models can take six measurements at a time; more advanced current models can measure 16
• Forward scatter: represents laser light scattered just slightly off-beam; intensity roughly proportional to size
• Side scatter (SSC): measured at about 90 degrees off-beam, and is high for cells with a lot of internal granules or segmented nuclei
• Remaining measurements are of laser-induced fluorescence - which we control by adding specific Abs tagged with fluorescent ligands that emit light, when hit with a laser, at characteristic wavelengths

Question 21

Be aware this is where blasts are usually run (this person has increased blasts).

Answer 21

Good job!

Question 22

Describe what you see here, and its implications. Can you tell what fraction of the bone marrow cells these cells represent?

Answer 22

• Myeloid blasts: express CD34 and 33
• While flow cytometry is great for characterizing blasts, it can’t be used to tell what fraction of bone marrow cells they represent -> have to rely on pathologist’s manual count for that
  1. In this case, 60% of total -> consistent with AML

Question 23

You suspect your patient has a hematologic malignancy involving a particular translocation, but cytogenetic studies are normal. What next?

Answer 23

FISH studies

Question 24

What if you can’t do flow cytometry?

Answer 24

• Do immunohistochemistry: you might want to do this if cells are too large for flow cytometry, or you want to see correlation between immunophenotype and morphology

-

Question 25

What does pathologist use to most accurately count blasts in bone marrow biopsy/aspirate?

Answer 25

• BONE MARROW ASPIRATE (because you want to avoid hemodilution)
• Number pathologist counts on slide is what is important for diagnostics

FROM GOOGLE:

• To ASPIRATE bone marrow fluid a needle is pushed through the anaesthetised skin into the bone. A syringe is used to draw out some liquid bone marrow. As the liquid is withdrawn, your may have a brief, sharp pain in your bone (and into the buttock and leg if the pelvic bone is used)
• To BIOPSY (tissue sample) bone marrow a second, thicker, hollow needle is inserted into the bone. This is rotated around as it is pushed slightly forward to force a small sample of bone marrow into the hollow middle of the needle. This may cause some dull pain for a short time. The needle is then taken out and a pressure bandage applied to prevent bleeding

Question 26

How do you determine the genotype of any abnormal cells in a marrow biopsy?

Answer 26

• Routine cytogenetics
• FISH
• DNA/RNA sequencing: targeted, PCR-based
• Complete genome or exome sequencing

Question 27

How do routine cytogenetic studies work?

Answer 27

• Growth medium: arrest of mitosis
• Squash cells onto a slide
• Optimal staining methods
• IDENTIFY and ENUMERATE the chromosomes present in dividing (metaphase) cells

Question 28

What is the Philadelphia chromosome?

Answer 28

• A translocation, or exchange of DNA, between chromosomes 9 and 22
• Specifically, between a region on the long (q) arm of 9 and the long (q) arm of 22

Question 29

How does FISH work?

Answer 29

• Method to visualize chromosomes in cells with intact nuclei (interphase cells: resting stage of cells) b/c not always easy to to do in metaphase cells (typical cytogenetics); cells do NOT have to be growing
• Process: 1) slides with unstained cells, 2) protease digestion to expose DNA, 3) fluorescent oligonucleotides specific for chosen targets (one color for each target)
• Results of a FISH study seen here: similar probes, or even combos of three, are now used
  1. NOTE: sometimes FISH will detect something you didn’t see in cytogenetics b/c you didn’t get good enough sample; if you had trouble getting good aspirate, but got good biopsy, FISH might work (or it might not)

Question 30

A clonal proliferation of megakaryocytes causing the platelet count in peripheral blood to triple would be an example of?

Answer 30

Myeloproliferative disease

Question 31

A recently diagnosed AML patient underwent targeted sequencing of multiple oncogenes and was found to have GOF mutations in Flt3. What kind of clinical trial might be best for him?

Answer 31

Tyrosine kinase inhibitor (TKI)

Question 32

What is DNA/RNA sequencing in this context?

Answer 32

• Process shown on image
• There are likely to be lots of genetic abnormalities that can’t be detected by either routine cytogenetic studies or FISH (the white space on the right)
• NOTE: based on this paper, sequence data for three oncogenes (NPM1, FLT3, and CEBPA) now REQUIRED in the evaluation of any AML patient whose routine cytogenetic studies are normal. To underscore that point: sequencing of targeted genes is an essential tool in the diagnosis of acute myeloid leukemias

Question 33

What is complete genome/exome sequencing?

Answer 33

• Comparison of neoplastic cells to the presumably unmutated DNA in each patient’s unaffected genome, usually obtained via a skin biopsy sample
• This is important because genomic sequencing and related technologies (exome sequencing, studies of epigenetic features like DNA methylation) make many more questions possible about how malignancies are generated and how we should diagnose, categorize, and treat them

Question 34

Is there a genetically relevant pattern to point muts, translocations, and deletions affecting specific genes, or groups of genes?

Answer 34

Yes

Question 35

Do genetic findings tell us something about pathogenesis?

Answer 35

Yes

Question 36

Does understanding pathogenesis open up some treatment options?

Answer 36

Yes

Question 37

What are the three major categories of malignancies arising in the bone marrow? Describe them.

Answer 37

• Acute leukemias: blasts (best #’d via morphology, but best identified by immunophenotype) proliferate in blood, bone marrow, or both
• Myeloproliferative diseases: similar clone (usually via cytogenetics and/or FISH studies) proliferates AND differentiates, yielding an increase in some type of peripheral blood cell
• Myelodysplasias, similar clone (usually evident via cytogenetics) proliferates and differentiates, but combo of the two processes does not result in normal blood cell production -> instead it yields (usually) a low count of one or more blood cells types (cytopenia(s)), usually in association with an abnormal appearance of those cell types (dyspoietic features)

Question 38

What is the clinical presentation of acute leukemias?

Answer 38

• Medical emergencies that can be associated with proliferation of blasts in # of locations
  1. Many blasts in blood and bone marrow (i.e, AML, aka myeloid sarcoma)
  2. Few blasts in blood, many in marrow
  3. Blasts outside marrow (rare)
• May also declare themselves as cytopenias, usually with leuko-erythroblastic findings on peripheral smear

Question 39

What is AML?

Answer 39

• Rapidly proliferating clones from one or more of the following:
  1. Myeloid lineage
  2. Erythroid lineage
  3. Megs

Question 40

What is ALL?

Answer 40

Proliferation of naive lymphos (blasts in marrow, and often, bloodstream)

Question 41

What is acute undifferentiated leukemia?

Answer 41

Rapidly proliferating clones derived from stem cells that have not yet committed to a lineage (this is all we need to know about these)

Question 42

>50% of childhood leukemias (ALL) have inactivating or dom negative mutations in one of which three transcription factors?

Answer 42

• IKZF1 (IKAROS)
• EBF1
• PAX5
• Impaired differentiation is a key feature of ALL

Question 43

What are the 3 major pathogenetic pathways at work in the development of AML?

Answer 43

1. Mutations that block maturation
2. Mutations that enhance proliferation
3. Mutations that cause genetic instability

Question 44

As a general rule, what do mutations that block differentiation look like?

Answer 44

• As a general rule, mutations that block differentiation alter large patterns of gene expression
• Include transcription factor fusions & some DNA methylation and chromatin modification mutations
• PML-RARA: in many cases, this is the ONLY coding sequence mutation detected. Even when o/muts are present, they are few in number -> PML-RARA translocation defines a distinct category of AML
  1. Distinctive clinical, pathologic features, and knowing pathogenetic mechanism allowed an effective therapy to be developed

Question 45

What kind of mutations enhance proliferation?

Answer 45

• Those that activate signaling initiated by EC ligands
• Important b/c in many cases the constitutively activated signal transduction molecules susceptible to targeted therapy
• In this group, tyrosine kinases (Flt3, Kit, and others) useful -> tyrosine kinase inhibitors designed to target specific kinases have been very effective in other contexts, i.e., CML
• However, some DNA methylation mutations appear to also enhance proliferation

Question 46

What is the significance of this image?

Answer 46

• Activation of proliferation-related genes leads to acute leukemias and other malignancies
• Mutants that lead to excess activity of proliferation-associated genes include those that activate certain tyrosine kinases (TK), particularly Flt3
• They also include those that reduce the activity of a methyl transferase (DNMT3A) that normally maintains high level of methyl cytosine modifications embedded somewhere in the relevant genes

Question 47

What is the significance of this image?

Answer 47

• Muts that lead to reduced activity of differentiation-associated genes include key translocations that generate activated derivatives of key transcription factors like RARA
• Tet1 and Tet2 mutations = persistent cytosine methylation of their target genes. These enzymes normally begin process of demethylating cytosine residues in certain target genes
• Their activity is localized to their target genes by WT-1 -> mutants in WT-1 have same effect on methylation patterns and same role in generating AML
• Tet1 and Tet2 need a cofactor, alpha keto-glutarate (from Kreb’s Cycle) to begin de-methylation process by oxidizing methyl groups to hydroxy-methyl groups. GOF mutant in enzyme that generates alpha-kg (isocitrate dehydrogenase, or IDH) = overproduction of molecule that’s very similar to alpha-kg (2-hydroxy-glutarate), and THAT molecule INHIBITS Tet1 and Tet2. Two IDH isoforms in which these muts are found
• Bottom line: mutants in all five of these genes (Tet1, Tet2, IDH1, IDH2, and WT-1) operate via SAME MECH, excess methylation of a set of differentiation-associated target genes

Question 48

What does the pathway to genetic instability typically look like?

Answer 48

• TP53: both alleles often disabled, and pts show high frequency of mutations in o/genes AND unfavorable cytogenetic findings
• Associated cytogenetic findings incl. translocations, deletions, or fragmentation, and correlate with a poor clinical outcome (hence the “unfavorable” category)

Question 49

Chromosomal translocations can contribute to a malignancy by inducing?

Answer 49

Overexpression of an oncogene

Question 50

What is the Knudson hypothesis? What other two aspects of p53 should we understand?

Answer 50

• Oncogenesis involving loss of both copies of the gene
• p53 normally functions to promote DNA repair by halting cell cycle until any necessary repairs are made.
• p53 ormally functions to induce apoptosis in cells that have suffered severe damage or are terminally senescent (makes some chemotherapeutic agents are effective)
• Knocking out both functions is very, very bad news, because it increases the number of mutant clones and makes it harder to kill them

Question 51

What is the significance of this chart?

Answer 51

• Genetic information from next gen sequencing is particularly promising in AML because these patients have relatively few “tier 1” mutations compared to other types of malignancy
• Good news – we have a smaller number of mechanisms to figure out and targets to hit with new or on-the-drawing-board therapies

Question 52

Which method can directly tell you the immunophenotype of abnormal cells in a bone marrow biopsy?

Answer 52

Flow cytometry