Hematologic Malignancies: Path & Dx Methods Flashcards
What is a hematologic malignancy?
A hematologic malignancy is an abnormal proliferation of cells derived from those normally found in the blood, bone marrow, or lymphatic tissues
What is a leukemia?
A hematologic malignancy that only involves the bone marrow and/or bloodstream.
What is acute leukemia?
When cells found in bone marrow, blood, or lymphatic tissues are found to be proliferating rapidly, they can be an immediate threat to the pt’s life. This is acute leukemia.
What are blasts, in the context of leukemia? (definition of ‘blast’ varies based on context)
The abnormal cells in cases of hematologic malignancy are usually called blasts because they are thought to represent a neoplastic transformation of rare, morphologically distinct precursor cells in the bone marrow that carry the same name.
What is chronic leukemia?
When the abnormal cells are proliferating slowly and are not an immediate threat to the patient’s life, the condition is called a chronic leukemia.
What is lymphoma/myeloid sarcoma?
When it only involves lymphatic tissue (lymph nodes, spleen, the subepithelium of the GI tract) or other sites, it is called a lymphoma or a myeloid sarcoma.
What is lymphoproliferative disease?
Abnormally proliferating lymphocytes can simultaneously involve the peripheral blood as well as lymphatic tissue. General terms in use for these conditions are lymphoproliferative disease or leukemia/lymphoma.
Describe how hematologic malignancies are Dx’d.
How hematologic malignancies are diagnosed:
Clinician recognizes a possible malignancy:
- Examples: Leukocytosis. Pancytopenia. Lymphadenopathy. Splenomegaly.
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.
Pathologist makes an initial assessment, orders confirmatory tests
- Examples: Flow cytometry. Immunohistochemistry. Cytogenetics. FISH. DNA sequence analysis.
5) Pathologist makes the diagnosis. Or not.
When should you suspect a hematologic malignancy?
1) When the bone marrow is not functioning normally, and you can’t find a simpler explanation.
Examples:
- Unexplained low cell counts (cytopenias; leukocytopenia; pancytopenia)
- Unexplained high cell counts (leukocytosis, erythrocytosis, thrombocytosis)
- Cells normally found in the bone marrow are present in the peripheral blood (leukoerythroblastic or myelophthisic features). This can include the presence of blasts.
2) When the lymphatic tissues have enlarged (lymphadenopathy, splenomegaly) and an infectious etiology can’t be found.
What is a blast? (based on morphology)
The most commonly accepted, gold standard definition is based on morphology:
It’s a cell with an appearance similar to that of undifferentiated hematologic precursor cells.
Features: 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
Is there an easy morphologic tip-off that the cell you’re looking at is a MYELOID blast?
Auer rods are needle like, eosinophilic (red) crystals formed by proteins normally found in the secondary (red) granules of granulocytes (chiefly myeloperoxidase).
Auer rods are always diagnostic of myeloid blasts.
The workup of any hematologic disease begins with a review of:
the peripheral smear
Blasts normally make up __% of the cells present in a bone marrow aspirate.
< 5%
In the bone marrow, myeloid cells (monocytes, and granulocytes) should outnumber erythroid precursors by __:__ or __:__, representing the majority in the bone marrow.
2:1 to 5:1
_____________ 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.
** Manual count **
When observing a core biopsy of bone marrow, describe what the normal cellularity looks like, based on age of the pt.
cellularity is 100-age
i.e. if the pt is 30, there should be 70% cellularity, the rest being fat and bone spicules.
When looking at a bone marrow core biopsy under the scope, you will observe myeloid precursors as (choose: light/dark) staining and erythroid precursors as (choose: light/dark) staining cells.
Myeloid: lighter staining
Erythroid: darker staining
What is the best way to gauge a pt’s Fe stores, just using a microscope? (according to Dr. Strom, this is the best method, period)
Prussian Blue stain of bone marrow core biopsy
In immunophenotyping, you can tell what type of cell you are looking at by:
using Abs specific for cell surface proteins that are only present on certain cell types.
ex: CD3, CD4, or CD8 on T cells
CD20 on B cells
The most reliable means of counting particular cell types in bone marrow aspirates is:
Why does this complicate the red cell precursor count?
Flow cytometry
Because the aspirate contains many red cells from the circulation (hemodilute: not representing actual contents of the bone marrow), there is a red cell lysis step that will also lyse the red cell precursors that were present in the bone marrow aspirate.
In flow cytometry:
“Forward scatter” represents laser light scattered just slightly off-beam, and its intensity is roughly proportional to:
cell size
In flow cytometry:
“Side scatter” (SSC), measured at about 90 degrees off-beam, is high for:
cells with a lot of internal granules or segmented nuclei
“complex cells”
In flow cytometry, remaining measurements besides forward scatter and side scatter indicate:
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.
This is how particular cell types are tagged (via Abs) and accounted for
Can flow cytometry currently be used to measure what fraction of bone marrow cells a sample represents?
No. But it is great for characterizing blasts!
In flow cytometry, when characterizing blasts, CD34 represents what kind of blasts?
hematopoietic stem cells (myeloid blasts)
In flow cytometry, when characterizing blasts, CD33 represents what kind of blasts?
granulocytes (myeloid blasts)
Describe routine cytogenetics as a method of genotyping abnormal cells.
Routine cytogenetic studies identify and enumerate the chromosomes present in dividing (metaphase) cells.
Describe fluorescent in situ hybridization (FISH) as a method of genotyping abnormal cells.
It’s not always easy to visualize chromosomes in metaphase cells. A method to do so in cells with intact nuclei (“interphase” cells) would be useful. FISH is that method. Key advantage to FISH is that the cells don’t have to be growing (as they do in conventional cytologic methods)
What diagnostic technique is essential in the Dx of acute myeloid leukemias (AML)?
sequencing of targeted genes
The ultimate method (from our current perspective) for genetically characterizing malignancies is:
to sequence them entirely – either the entire genome (in the range of 3 billion nucleotides) or the exome. This requires comparison, of course, to the presumably unmutated DNA in each patient’s unaffected genome – usually obtained via a skin biopsy sample.
What can you learn from complete genome sequencing?
There is a clinically relevant pattern to point mutations/translocations/deletions affecting specific genes or groups of genes.
The genetic findings tell us things about pathogenesis
- understanding the pathogenesis opens up treatment options
Of the following:
Myeloproliferative disease
Acute leukemia
Myelodysplastic syndrome (MDS)
Which is associated with rapidly proliferating clones:
Blasts in marrow and, often, bloodstream?
Acute leukemia
Of the following:
Myeloproliferative disease
Acute leukemia
Myelodysplastic syndrome (MDS)
Which is associated with chronically proliferating clones which differentiate to circulating blood cells?
myeloproliferative disease
Of the following:
Myeloproliferative disease
Acute leukemia
Myelodysplastic syndrome (MDS)
Which is associated with poorly functioning clones?
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)
Myelodysplastic syndrome (MDS)
Describe the 3 clinical presentations of acute leukemias with regards to where the malignancies are found. (where are there larger than normal numbers of cells?)
Can be:
Many blasts in blood and marrow
Few blasts in blood, many in marrow
Blasts outside marrow (rare)
- 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.
Untrue to the name, acute myeloid leukemias are defined as rapidly dividing clones from any of these three lineages:
Myeloid (monos, neutros, eos, basos), erythroid, megakaryocytes.
(pretty much anything but lymphocytes)
Acute lymphoblastic leukemia involves this cell line:
lymphocytes
What is an acute undifferentiated leukemia?
clones that appear to be derived from stem cells which have not committed to a lineage (very rare)
Describe three major pathogenetic pathways that are at work in acute myelogenic leukemia. (mutations that cause malignancy)
1) Mutations that block maturation
2) Mutations that enhance proliferation
3) Mutations that cause genetic instability
With regards to AML:
As a general rule, how do mutations blocking differentiation work? What about the DNA changes?
They alter large patterns of gene expression.
They include transcription factor fusions as well as some of the DNA methylation and chromatin modifications.
With regards to AML:
One particular transcription factor gene, ________, is the ONLY coding sequence mutation detected that results in malignancy.
RARA fusion with PML (PML-RARA)
With regards to AML:
How do mutations that enhance proliferation work? What cell pathways do they alter?
They activate signaling initiated by extracellular ligands. These specific, constitutively activated signal transduction molecules are susceptible to targeted therapy.
Some DNA methylation mutations also appear to enhance proliferation.
What are Flt3 and DNMT3A and why are they important to acute leukemias and other malignancies?
Flt3 is a tyrosine kinase that is constitutively activated in some acute leukemias and other malignancies.
DNMT3A is a methyl transferase that normally maintains a high level of methyl cytosine modifications embedded somewhere in relevant genes that keeps them turn’t off.
What are Tet1 and Tet2 and why are they relevant to blood cancers? Do they lead to increased or decreased differentiation?
Mutations that result in persistent cytosine methylation of their target genes.
Lead to decreased differentiation.
The unmutated genes code for enzymes that normally begin the process of demethylating cytosine residues in certain target genes.
What is WT-1 and why is it significant to blood cancers?
WT-1 localizes Tet1 and Tet2 to their targeted genes. Mutations in WT-1 cause Tet1 and Tet2 to not demethylate their targeted genes, resulting in persistently methylated cytosine residues that normally signal for cell differentiation. This leads to undifferentiated cells (aplasia bitches!)
Why is alpha-ketoglutarate relevant to blood cancers? Wasn’t that shit in the Krebs Cycle?
a-ketoglutarate is a cofactor for Tet1/2 and is needed to begin demethylation.
It turns out that a gain-of-function mutant in the enzyme that generates alpha-kg (isocitrate dehydrogenase, or IDH) results in overproduction of a molecule that’s very similar to alpha-kg (2-hydroxy-glutarate), and THAT molecule INHIBITS Tet1 and Tet2. So cells won’t differentiate. (aplasia bitches!)
So big picture, mutations in Tet1/2, Wt-1, IDH1/2, all operate via this mechanism in causing cells to remain undifferentiated:
Allow for excess methylation (because those genes are needed to demethylate differentiation genes). Thus, NO DIFFERENTIATION. (APLASIA BITCHES)
Why is TP53 important to blood cancer?
Double hits to TP53 (fully knocking out the TSG) results in genetic instability that leads to more cancer cells that are even more difficult to kill because they refuse to undergo apoptosis or senescence.
50% of childhood acute lymphoblastic leukemia (ALL) cases have inactivating of dominant negative mutations in one of these three transcription factors:
Also list the step of maturation that is inhibited.
IKZF1 (Stem cell -> lymphoid precursor)
EBF1 (Pro-B -> Pre B1)
PAX5 (Pre B1 -> Pre B2)
Which of these methods can directly tell you the immunophenotype of abnormal cells in a bone marrow biopsy? FISH studies Conventional cytogenetics Flow cytometry targeted DNA sequencing A differential count
Flow cytometry
You suspect your pt has a hematologic malignancy involving a particular translocation, but his cytogenetic studies are normal. Among the following options, what's the best way to follow up? FISH studies flow cytometry immunohistochemistry bone marrow differential count whole exome sequencing
FISH studies
A clonal proliferation of megakaryocytes causing the platelet count in peripheral blood to triple would be an example of which of the following? Myelodysplastic syndrome Myeloproliferative disease Acute myeloid leukemia Acute undifferentiated leukemia It depends on the cytogenetic findings
Myeloproliferative disease
A recently Dx’d AML pt underwent targeted sequencing of multiple oncogenes and was found to have gain of function mutations in FLT3. What kind of clinical trial might be best for him of the options listed?
A tyrosine kinase inhibitor
A drug that inhibits cytosine methylation
A drug that induces apoptosis
A monoclonal Ab targeting CD20
DNA methylation
A tyrosine kinase inhibitor
A recently Dx’d AML pt underwent targeted sequencing of multiple oncogenes and was found to have gain of function mutations in IDH1. What kind of clinical trial might be best for him of the options listed?
A tyrosine kinase inhibitor
A drug that inhibits cytosine methylation
A drug that induces apoptosis
A monoclonal Ab targeting CD20
DNA methylation
A drug that inhibits cytosine methylation
Demethylation of genes is dependent on actions by Tet1 & Tet2 and IDH1 & IDH2
If Tet1/2 & IDH1/2 mutated, genes will remain methylated and will not be expressed to induce differentiation.
Chromosomal translocations can contribute to a malignancy by inducing: hyperdiploidy point mutations overexpression of an oncogene gene amplification DNA methylation
overexpression of an oncogene
