Transcript Hematology and Hematological Malignancies

Hematology and Hematologic
Malignancies
Cancer of the formed elements of
the blood
What is hematology?


Hematology is the study of blood and is
concerned mainly with the formed elements
in the blood.
The formed elements in the blood include:

The white blood cells (leukocytes) which include
the





myeloid cells: neutrophils, eosinophils, basophils,
monocytes
lymphocytes.
The red blood cells (erythrocytes)
The platelets (thrombocytes)
All of the formed elements in the blood are
derived from same pluripotential stem cell in
the bone marrow
What is hematology? (cont’d.)



Erythrocytes (red blood cells) function
in the transport of oxygen to the
tissues.
Leukocytes (white blood cells) function
in both specific (immune responses)
and non-specific defenses against
foreign invasion.
Thrombocytes function in hemostasis or
blood clotting.
What is a hematological
malignancy?

A hematologic malignancy is a
malignancy (or cancer) of any of the
formed elements in the blood.
What is a hematological
malignancy?

The malignancies may be classified into

Lymphomas



Lymphoid (or lymphocytic) leukemias





Hodgkins versus non-Hodgkins
B cell versus T cell
Chronic versus acute
B cell versus T cell
Acute myelogenous leukemia
Myelodysplastic syndromes
Myeloproliferative disorders (includes
chronic myelogenous leukemias)
What is a hematological
malignancy (continued)

For the purposes of this class we will concentrate
only on the hematological malignancies of
lymphocytes, i.e., lymphomas and lymphocytic
leukemias.

Remember that the malignant cell in a leukemia
originates in the bone marrow and that the malignant
call in a lymphoma originates in tissue other than the
bone marrow.

(The word myeloid or the prefix myelo- refers to cells that are
not lymphoid or lymphocytic (e.g., neutrophils, eosinophils,
basophils; not T cells and B cells).
What is a hematological
malignancy (continued)


The lymphoid malignancies are a
heterogeneous group of disorders that
occur as a result of neoplastic
transformation at different stages of B cell
and T cell development.
A lymphoid malignancy may develop at any
stage of development of B or T cells.
Notice the cell and stage-specific markers on this and the subsequent slide.
The maturity of the cell at the time the cancer developed can be determined by
doing a molecular assay for which Ig (BCR) genes have rearranged.
What are these various cell
surface “CD” markers?




All cells carry various functional proteins in their
membranes which are at least partly exposed on
the cell surface.
CD molecules are cell surface molecules expressed
on various cell types in the immune system.
They are designated by the “cluster of
differentiation” or CD nomenclature, which
includes “CD” followed by a number, as in CD4.
The various CD molecules and their known or
proposed functions are listed in Abbas and
Lichtman “Basic Immunology.”
The maturity of the cell at the time the cancer developed can be determined by
doing a molecular assay for which TCR genes have rearranged.
Some abbreviations from previous slides





ALL – acute lymphocytic leukemia
CLL – chronic lymphocytic leukemia
CTCL – cutaneous T cell lymphoma
PTCL – peripheral T cell lymphoma
LGL – larger granular lymphocytic leukemia
Determining the origins and
classifying B and T cell malignancies

Cancers arising from cells that are already
immunocompetent or that are undergoing antigenindependent maturation will have characteristic
rearrangements of their B cell receptor (Ig) or T cell
receptor genes.



The DNA is examined to determine whether or not a rearrangment
has occurred, and whether it is in a B cell or T cell gene
This method is very accurate.
In cytopathology, the level of cellular differentiation is used
as a measure of cancer progression. “Grade” is a marker
of how differentiated a cell in a tumor is. Often the less
differentiated, the more readily cell division takes place.

Cell surface markers of maturity on lymphocytic cells are more likely
to be accurate early in the development of the cancer. (Sharp’s
best guess.)
What are the criteria for classification as a
malignant proliferation of hematopoietic
cells?

Monoclonality


Clonal progression –


all malignant cells arose from a single cell
once started the proliferation does not stop, i.e.
the malignant clone expands
Clonal dominance –

a proliferative advantage allows the malignant
clone to replace normal cells and their
descendants


Grows faster
Secretes something that interferes with expansion of
normal clones
Criteria for Classification
continued


Extinction of normal clones – early in disease
progression, normal clones are present, but
suppressed. Later in the disease progression,
all cells of normal clones die.
Genetic instability – as the malignant clone
proliferates, subclones arise with properties
less and less like normal cells
(well differentiatedless differentiated)
What is cancer?

Cancer is a form of genetic disease.





This does not mean that you can inherit cancer.
Cancer is the result of a multistep process.
Cancer is characterized by an accumulation of
multiple genetic mutations in a population of
cells undergoing neoplastic transformation.
After the first mutation, there is limited
expansion
After subsequent mutations, there is greater
proliferation potential.
How does this happen?
In the following slides:
= a non- dividing cell
1, 2, 3 = successive
mutations, each contributing
in some way to an increased
rate of cell division or
decreased rate of cell death.
1
Non-dividing
cells
1
1
Non-dividing
cells
2
1
1
1
1
2
2
1
32
2
Non-dividing
cells
Non-dividing cells
1
1
12
12
1
123
12
12
123
123
123
123
123
123
123
123
This process continues, with each successive mutation
leading to a faster rate of cell division, slower rate of cell
death, and eventually loss of cell adhesion.
A sort of summary of
the previous four
slides.
What is cancer, continued

The progression of cancer is easily
documented in some tumor systems:




Benign – tumor is not recurrent
Malignant – the tumor tends to become
progressively worse
Metastatic – the tumor is capable of spread to
distant sites
With hematological malignancies, the
progression is often not as obvious.
Causes of Change
Features
Why is it important to determine the cell
lineage of a leukemia or a lymphoma?


Different types of leukemia and
lymphoma are treated with different
types of chemotherapy.
Different types of leukemia and
lymphoma have different prognoses.
How do you determine the cell
lineage of a leukemia or lymphoma?


Morphologic (structural) characteristics of the
malignant cells (done by a pathologist)
Cytochemistry (stain for the presence of
specific enzymes or lipids and glycogen
associated with specific types of cells)




Myeloperoxidase
Esterase
Sudan black
Terminal deoxynucleotidyl transferase
How do you determine the cell
lineage of a leukemia or lymphoma?

Immunohistochemistry (look for the presence of cell
surface markers with antibody probes)




Immunoglobulin
CD4
CD8
Molecular tests for rearrangement of the T or B cell
receptor (antibody) gene
How do you diagnose a leukemia or
lymphoma?


Cytology – microscopic examination
Cytogenetics (chromosome analysis, chromosome
painting)


Chromosomal translocations
Molecular tests


Restriction digest of genomic DNA+Southern blotting
PCR+/- Southern blotting of PCR products

PCR for chromosomal translocations and overgrowth of any
monoclonal lymphocyte population.
Molecular tests are expensive. Why would
one use a molecular test for the diagnosis
of a hematological malignancy?


To prove that a malignancy is present
when the cells do not appear
morphologically malignant.
To prove that a neoplastic population of
B or T cells is monoclonal in origin


If the entire population is from one clone,
it is in late stage (see earlier slides)
To look for chromosomal translocations
How can one prove that a neoplastic
population of cells is monoclonal in
origin?


Southern blot without PCR
PCR (we will only discuss this method which
has many advantages over Southern blotting)




Cheaper and quicker
Requires less initial DNA
DNA can be of low quality
Can detect a monoclonal population that
comprises as little as 0.1% of the total population
of cells (as compared to 5% for the Southern blot
without PCR)
How is PCR used to establish the presence of a
monoclonal population of malignant cells?
Note: this approach works even when there is
no translocation involved in the development
of the hematological malignancy.
How is PCR used to establish the presence of a
monoclonal population of malignant cells?


Isolate or extract DNA (biopsy, bone
marrow)
Next slide
PCR to identify a
monoclonal population
of cells
How is PCR used to establish the presence of a
monoclonal population of malignant cells?
(cont’d.)

PCR using consensus primer pairs (i.e., primer
pairs that recognize all V or J segments) for the
immunoglobulin heavy chain (for monoclonal B
cells ) or TCR (for monoclonal T cells).


In the germline DNA these primers are too far apart to
give a good PCR result, because rearrangement has not
occurred.
The only cells in which a PCR product will be generated
are cells in which a DNA rearrangement has occurred to
bring the V and J segments close enough to generate a
PCR product using the consesus primers
How is PCR used to establish the presence of a
monoclonal population of malignant cells?
(cont’d.)

Run the PCR products on an agarose gel

Remember that DNA rearrangement is a normal
process that occurs during the normal
maturation of B and T cells to
immunocompetent B and T cells.
How is PCR used to establish the
presence of a monoclonal population
of malignant cells?


From a normal individual, there should be
fragments of many different lengths – which will
look like a smear of DNA from the many different
VDJ rearrangements (a normal polyclonal
population of cells)
If an individual has an abnormal expanded
monoclonal population of cells, a distinct band will
be seen, even when the monoclonal subpopulation
of cells is as low as 0.1% of the total population of
cells.
PCR to identify a
monoclonal population
of cells
Remember


Monoclonal malignancies may or may not
involve a translocation as one of the
mutations.
When a chromosomal translocation is
involved in either leukemia or lymphoma, a
proto-oncogene and a portion of an IgG gene
are often transposed from one chromosome
to another and located next to each other.
Review



We have used PCR to detect a
translocation.
This lecture introduced use of PCR to
detect a monoclonal lymphoma or
leukemia that did not necessarily involve a
translocation.
What are the technical and biological
differences?
How is PCR used to identify a
chromosomal translocation?



Perform PCR on patient tumor DNA using
consensus primers. One primer will bind to a
region on one chromosome while the other
primer will bind to a region on the other
chromosome.
There is no product if a translocation between
the two chromosomes has not occurred.
Confirm with specific probe.
Answer to naming leukemias and
lymphomas

From: Wilczynski, Sharon
[mailto:[email protected]]
Sent: Wed 5/20/2009 4:30 PM
To: Glackin, Carlotta
Cc: Sharp, Sandra
Subject: RE: Question about cancer
The hematopathologists have made it their life work to classify and then reclassify and change the
names of theses cancers. It is a special club and only they seem to understand the
nuances. However, as a general surgical pathologist, I can tell you that site the tumor arises in is
not really that important although historically it was used. Now we are tending to classified by the
cell of origin (usually immunohistochemistry is used to identify the lineage of the cell) but more and
more we are looking at the underlying molecular defect.
Lymphoma is a a neoplastic proliferation of lymphoid cells arise in lymphoid tissue anywhere in the
body (usually that is lymph node but it can be organs like stomach or cervix or in the bone
(osteolymphoma). These are B-cell lymphomas (origin from precursors to immunoglobulin
secreting cells and will have gene rearrangments of the immunoglobulin heavy and/or light
chains). T-cell lymphomas have rearrangments of the T-cell receptors.
Leukemia is from the hematopietic cells (precursors to granulocytes or polymorphonuclear
leukocytes, red blood cells, or platelets). But it isn't that straight forward. To confuse the issue
you do have acute lymphoblastic leukemia (ALL) which usually presents with elevated blasts in
blood and is considered a leukemia, Sometimes it presents with a mass and then it is termed
lymphoblastic lymphoma. There is also chronic lymphocytic leukemia/small lymphocytic
lymphoma which are really the same entity but now are classified as B-lymphoblastic
leukemia/lymphoma and then characterized by the type of translocation such as (9:22). As we
learn more of the molecular we are splitting and combining diagnosis. For example the EWS/FLI-1
translocation has united into one diagnostic category Ewings sarcoma and peripheral primitive
neurectodermal tumor.
With the leukemias and lymphomas the underlying molecular fundamental alteration is altering how
we name them. I would recommend that you use the World Health Organization Classification for
leukemia and lymphomas. They just redid it last year again. If you don't have acess to it I can
send you the short classification scheme. Sharon