Hematologic Malignancies

Question 1

When a hematological malignancy involves the bone marrow and/or bloodstream, what is it called?

Answer 1

a leukemia

Question 2

When these cells are proliferating rapidly, they can be an immediate threat to the patient’s life it’s called?

Answer 2

acute leukemia. Less aggressive proliferations are called chronic leukemias.

Question 3

Acute leukemias can be further divided into:

Answer 3

• myeloblastic
• lympholoblastic
• lymphoma (aka myeloid sarcoma)

based on cell line

Question 4

What is an indolent lymphoma?

Answer 4

aka low grade lymphoma

when the abnormal cells involving lymphatic tissues are proliferating slowly and are not an immediate threat to the patient’s life

aggressive= high grade lymphoma

Question 5

What is a lymphoproliferative disease?

Answer 5

when abnormally proliferating lymphocytes simultaneously involve both peripheral blood and lymphatic tissue

Question 6

What is Depth of sequencing?

Answer 6

refers to the number of times, on average, a given stretch of DNA is sequenced. That’s critical because tumor samples almost always contain more than one genotype (due to, for example, clonal evolution).

The 2008 AML study barely covered every region once. The 2013 study (and many subsequent studies) covered each region at least 30 times (in the cases studied via WGS) or over 70 times (via WES). And that has the potential to transform the study of cancer genetics from characterization of individual tumor types to the study of how malignant clones evolve within each tumor type – and each patient. That’s one way in which this field is likely to change hugely over the next decade.

Question 7

What are the major categories of malignancies arising in the bone marrow?

Answer 7

• acute leukemia
• myeloproliferative disease
• myelodysplastic syndrome

Question 8

What are acute leukemias?

Answer 8

conditions in which clonal BLASTS with features similar to those of hematopoietic stem cells proliferate in blood, bone marrow, or both.

KEY: these cells have acquired some combination of mutations that enhance proliferation and inhibit differentiation.

Question 9

What are myeloproliferative diseases?

Answer 9

a similar clone proliferates AND differentiates, yielding an increase in some type of peripheral blood cell.

Question 10

What are myelodysplastic diseases?

Answer 10

In myelodysplasias, a similar clone proliferates and differentiates, but the combination 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 11

The classic case of acute leukemia is what?

Answer 11

rapid growth in the bloodstream, and these usually also take over the bone marrow.

Sometimes, however, they will take over the bone marrow without raising the white cell count in peripheral blood.

Question 12

How else may acute leukemias present?

Answer 12

They may in fact declare themselves clinically as cytopenias, usually with the clues we call leuko-erythroblastic findings on the peripheral blood smear.

In some cases they present as a proliferation of immature cells outside both locations. This is particularly the case for acute myeloid leukemias with monocyte-like features, which can present clinically as solid tumors in bone or connective tissue. (That makes sense, since monocytes are mostly just precursors to tissue-based macrophages.)

Because malignancies arising in these affected tissues are usually termed “sarcomas”, the confusing older terminology for these masses of myeloid blasts (as noted earlier) is “myeloid sarcoma”.

Question 13

Clinical signs of acute leukemia:

Answer 13

• many blasts in blood and marrow
• few blasts in blood, many in marrow

or

• blasts outside marrow (rare)

Question 14

What are some hematopoietic stem cells?

Answer 14

• myeloid lineage (most common in marrow)
• granulocyte and monocyte precursors
• erythroid lineage
• megs
• lymphocytes

Question 15

How is AML (acute myeloid leukemia) defined?

Answer 15

proliferation of any of these lines:

• myeloid lineage
• erythroid lineage
• megs

not just myeloid lineage

Question 16

How is ALL (acute lymphoblastic leukemia) defined?

Answer 16

proliferation of just the lymphocyte lineage

Question 17

What is an acute undifferentiated leukemia?

Answer 17

clones derived from stem cells which have not committed to a lineage

Question 18

What are the mains steps of diagnosing a hematologic malignancy?

Answer 18

1) Clinician recognizes a possible malignancy:
- Examples: Leukocytosis. Pancytopenia. Lymphadenopathy. Splenomegaly.

2) Clinician requests/performs appropriate initial tests
- Examples: CBC, peripheral smear review, imaging studies

3) Clinician obtains tissue for pathologic confirmation of diagnosis
- Examples: Peripheral blood. Bone marrow biopsy/aspirate. Lymph node biopsy.

4) Pathologist makes an initial assessment, orders confirmatory tests
- Examples: Flow cytometry. Immunohistochemistry. Cytogenetics. FISH. DNA sequence analysis.

5) Pathologist makes the diagnosis. Or not.

Question 19

What things would lead you to suspect acute leukemia?

Answer 19

• The white cell count is 120,000 per ul (20 times normal)
• The lab calls you to say they are all blasts
• The patient was well until last week

this is not the diagnostic order, just an extremely obvious case

Question 20

Could the white cell count be normal in acute leukemia?

Answer 20

Yes

Question 21

When should you suspect a hematologic malignancy? Option 1

Answer 21

1) When the bone marrow is not functioning normally, and you can’t find a simpler explanation.

Question 22

What would be some clues that the bone marrow is not functionally properly?

Answer 22

• unexplained low cell counts (cytopenias, leukcytopenia, pancytopenia)
• unexplained high cell counts (leukocytosis, erythrocytosis, thrombocytosis)

Question 23

When should you suspect a hematologic malignancy? Option 2

Answer 23

When the lymphatic tissues have enlarged (lymphadenopathy, splenomegaly) and an infectious etiology can’t be found.

Question 24

What morphologic features suggest that a leukocyte is a blast? 5 things

Answer 24

• Large cells
• High nuclear/cytoplasmic ratio
• Prominent, single or multiple nucleoli
• Immature (faint/smudgy) chromatin
• Their appearance is shared by many cells on a slide

In most clinical settings, cells that we call blasts may only show three or four of these characteristics. The most important of them is number five, the “cookie cutter” appearance.

Question 25

Is there an easy morphologic tipoff that the cell you’re looking at is a blast (specifically AML)?

Answer 25

Auer rods

Question 26

What are Auer rods?

Answer 26

needle-like, eosinophilic (red) crystals formed by proteins normally found in the secondary granules of granulocytes (chiefly MPO).

They are alway diagnostic of myeloid blasts.

Question 27

The workup of any hematologic disease begins with what?

Answer 27

a review of the peripheral smear.

Question 28

What are some examples of cells normally found in the bone marrow that can be present in the peripheral blood (leukoerythroblastic or myelophthisic features) and is suggestive of bone marrow pathology?

Answer 28

• nucleated red cells
• basophilic stippling (non-specific) or Howell-Jolly bodies
• myelocyte
• blast
• giant platelets

In general, any two or three of these findings suggests bone marrow pathology - but does not tell you what TYPE of pathology is present. Unless the patient already has known bone marrow pathology (such as a metastatic carcinoma), these findings indicate a need to find out what’s going on in the bone marrow – via biopsy.

Question 29

What are Howell-Jolly bodies?

Answer 29

nuclear fragments that normally get yanked out of any circulating red cells by phagocytes in places like the spleen

They are seen when abnormal erythroid maturation is taking place, or when the spleen is absent.

Question 30

To find out what kind of pathology is present, you need to get bone marrow contents to a pathologist. How is this done?

Answer 30

Preps a sterile working field (usually posterior iliac crest)

Injects a local anaesthetic

Drives a large needle into the bone

Draws out (aspirates) a half cc of thick bloody fluid containing bony spicules
	-  Hands it to pathology

Draws out an additional 5-20 cc for special studies as needed
- Hands it to pathology

Inserts a larger needle into a different site or at a very different angle

Draws out a “core” biopsy within that needle
- Hands it to pathology

Question 31

What is the the “special studies” tube in most cases diluted with?

Answer 31

peripheral blood (“hemodilute”)

Question 32

Why would a core biopsy be taken at a different spot?

Answer 32

that the “core” biopsy will be empty of blood-forming elements if you extract it from exactly the same site where the aspirate was obtained.

Question 33

What does a pathologist do with all the things just collected by the physician?

Answer 33

A) Spreads the initial (0.5 cc) aspirate onto several slides; stains them the same way peripheral blood is stained
B) Puts some of the extra aspirated material clot in a tube, lets it clot
C) Sends the clot and the core biopsy for fixation, paraffin embedding, histologic sections
D) Sends off all special studies imaginable
OR
D) Takes an initial look at an aspirate slide under the scope
- Refines the differential diagnosis
- Orders appropriate tests
E) Reviews the routinely stained aspirate, core biopsy, clot sections
F) Orders other tests as appropriate
G) Pulls all the test results together into one report
H) Makes a diagnosis. Or not.

Question 34

What does a pathologist do later on?

Answer 34

A) Describes what any abnormal cells look like

B) Describes the immunophenotype any abnormal cells

C) Describes the genotype of any abnormal cells

D) Makes a diagnosis. Or not.

Question 35

What will the pathologist do with the aspirate slides?

Answer 35

The pathologist will count the different cell types present (generating the “differential count”.) Best for accurately counting marrow BLASTS

This manual count of the cell types in the bone marrow aspirate is currently the “gold standard” for determining whether there is an abnormal proliferation of blasts or not. Read that sentence one more time.

Question 36

Blasts normally make up less than __ of the cells present,

Answer 36

5%

and myeloid cells (granulocytes, plus monocytes) should outnumber erythroid precursors by 2:1 to 5:1.

Question 37

How long should the core biopsy be?

Answer 37

2-3 cm long

Question 38

Eqn for estimating cellularity of the core biopsy?

Answer 38

100-age= cell percentage. Rest=fat

i.e. at 40, you are 60% cell, 40% fat

Question 39

T or F. A good core biopsy will show well represented erythroid, myeloid, and megakaryocyte lineages.

Answer 39

T. Blasts are present, but in most cases they are hard to count in the core biopsy.

Question 40

What else could you see in the marrow?

Answer 40

Iron. Cyanide stains them blue, routinely, and the blue compounds are usually visible in both the storage cells (macrophages) and in some of the red cell precursors.

Question 41

What are some surface antigens on B cells?

Answer 41

• CD 45, CD79a, CD20

- IgG kappa or lambda

Question 42

What are some surface antigens on T cells?

Answer 42

• CD 45, CD 7, CD 4/8

- TcR (CD3)

Question 43

The most reliable means of counting particular cell types in bone marrow aspirates is what?

Answer 43

flow cytometry

but bear two things in mind: the aspirate used for this is usually hemodlute, and the red cell lysis procedure lyses most or all of the erythroid precursors. That’s why the morphologic count (by a pathologist, using a microscope) is still important.

Question 44

Describe the flow cytometry procedure.

Answer 44

Cells are collected from bone marrow (aspirate or other source), the red cells are lysed, fluorescent antibodies are added to desired cell surface proteins and the fluid scattered. So no red cells enter the cytometer

Question 45

How does the cytometer work?

Answer 45

The cytometer takes several optical measurements from each cell that passes though its “flow cell”, and can do this for dozens of cells per second. Most models in use can take six measurements at a time; more advanced current models can measure 16.

Question 46

What is forward scatter? Side scatter?

Answer 46

“Forward scatter” represents laser light scattered just slightly off-beam, and its intensity is roughly proportional to cell size.

“Side scatter” (SSC), measured at about 90 degrees off-beam, is high for cells with a lot of internal granules or segmented nuclei. The remaining measurements are of laser-induced fluorescence - which we control by adding specific antibodies tagged with fluorescent ligands that emit light, when hit with a laser, at characteristic wavelengths.

Question 47

The result is a huge amount of data from any given patient. How do we begin to analyze it?

Answer 47

Most hematopathology labs start by plotting CD45 expression (it’s on almost all marrow cells, but at different levels) vs. side scatter (SSC). Each dot on the plot represents two measured values taken from a single cell. You find the data falling into clusters which define the major cell types seen in bone marrow.

Note again that red cell precursors are under-represented due to the red cell lysis procedure; flow cytometry is for this reason a weak method for characterizing these cells.

Question 48

What surface antigens do blasts expres?

Answer 48

CD34, a marker of hematopoietic stem cells, and CD33, a marker of granulocytes

These features identify them as myeloid blasts.

Question 49

T or F. Flow cytometry is great for characterizing blasts, but it cannot (currently) be used to tell you what fraction of the bone marrow cells they represent.

Answer 49

T, You have to rely on the pathologist’s manual count (from aspirate slides) for that.

Question 50

What are other limitations of flow cytometry?

Answer 50

• It doesn’t work well for unusually large cells
• it can’t be immediately correlated with detailed morphologic features (like, for example, what other cells are near the cells of interest). Antibodies that mark cells on a slide can be used to provide additional immunophenotypic data in such cases.

Here, a large cell characteristic of Hodgkin’s lymphoma (the famous Reed-Sternberg cell) is identified by the combination of morphology and immunophenotype.