About the Philadelphia Chromosome

The Philadelphia chromosome is an acquired abnormality and is the definitive marker for CML.

It is formed when chromosomes 9 and 22 swop one part each with the other. It is not yet understood why this
happens. CML is not an inherited condition and as such cannot be passed on to children.

Known as a reciprocal translocation, a piece containing the ABL1 gene from the bottom part ofchromosome 9
breaks off and attaches to a region on chromosome 22 where the BCR gene is located, thus forming a
new chromosome 22 containing the abnormal fusion gene BCR-ABL1

The new shortened chromosome 22 is called the Philadelphia chromosome. You might also see it written as t(9;
22). The BCR-ABL1. fusion gene makes a protein called a tyrosine kinase. These kinds of proteinsare located on
or near the surface of cells and send signals that speed up cell division.

Ph+CML is a rare disease with an incidence of 1 to 1.5 new cases per 100.000 people each year. It is very rare
in young people under 19 and ultra-rare in young children.

In approximately 95% of cases, the Ph chromosome is detected by cytogenetic analysis. In around 5% of the
remaining suspected cases, the Ph chromosome is not visible. However, the BCR-ABL1fusion gene can be
identified by molecular testing with q-PCR in approximately half of those cases. For simplicity, any disease that
contains the BCR-ABL1 fusion gene is referred to as Ph+ CML. If you do not have the Ph chromosome or
evidence of the BCR-ABL1 gene then you do not have CML.

It has been shown that the BCR-ABL1 fusion gene is the single definitive marker for Ph+ CML and remains the
key abnormality throughout the chronic phase of the disease.

The BCR-ABL1 fusion gene translates as a protein tyrosine kinase, referred to as Bcr-Abl1. The activity
oftyrosine kinases is typically controlled by other molecules, but the mutant tyrosine kinase produced by
the BCR-ABL1 gene means the signal for cell division is always ‘switched on’. Its continuous activity sets up a
cascade of events that allows for unregulated cell division (cancer). Over time the Ph+ cells overpopulate the
marrow, crowding out normally functioning cells.
Since the introduction of targeted therapy with tyrosine kinase inhibitors (TKIs), it is increasingly evident that,
at least in chronic phase, when cells containing the BCR-ABL1 gene are reduced to very low levels, the risk of
disease progression is significantly reduced.

Genes are denoted with italicized capitals as in ABL1; BCR etc., whereas proteins are denoted with non-
italicized capitals, sometimes followed by lower case, as in Bcr-Abl1.

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Slide #DMS 104 [Marrow smear, Wright Stain].

This is a low power view of a marrow smear, the preferred preparation for examining the morphology of hematopoietic
cells. The cells streaming away from the small clots are often of good morphological quality and will be examined in
subsequent images.

A medium power view of a marrow smear shows a small clot surrounded by individually-distinguishable marrow cells, the
details of which are really best appreciated with 100x oil immersion optics. At this power, only the large, multilobulated
megakaryocytes can be unequivocally identified.
This oil immersion image shows the characteristic features of the early stages of erythroid development. Using mature red
blood cells as a yardstick, note the size of their developing precursors, beginning with the proerythroblast. This large cell
is characterized by a large, round nucleus containing one or more nucleoli (lightly stained). The cytoplasm of this cell may
show an intense royal blue basophilia. As development proceeds, these cells will become smaller, the nucleoli will
disappear, but the royal blue basophilic cytoplasm will persist into the basophilic erythroblast stage. Remember that
hematopoiesis represents a developmental continuum, and that many of the cells seen will be somewhere in between the
classically-defined stages.

Compare the size, nuclear morphology and cytoplasmic staining of the basophilic erythroblast (baso) with subsequent
stages of development, the polychromatophilic erythroblast (poly) and then the orthochromatophilic erythroblast (ortho).
The polys are characterized by a grayish-blue cytoplasm, and a round nucleus with a 'checkerboard' chromatin pattern.
With further development, these cells continue to shrink, the nucleus continues to condense, and the increased production
of hemoglobin gives the cytoplasm of the orthochromatophilic erythroblasts a faintly pinkish hue (but compare to mature
RBC).
In this image, note the changes in size, degree of nuclear condensation, and cytoplasmic coloration as one progresses
from polychromatophilic erythroblasts to the orthochromatophilic erythroblasts.

In this image, note the two orthochromatophilic erythroblasts, with rather eosinophilic cytoplasm (still not yet of the mature
hue, however) and dark, condensed nuclei. After the extrusion of their nuclei, these cells will then be called reticulocytes
prior to their final maturation into erythrocytes.
This image illustrates the feature of two promyelocytes, one of the early stages in the development of the granulocytes.
These large cells are characterized by multiple nucleoli, and a lightly basophilic cytoplasm in which one may find rather
large, azurophilic granules (aka primary granules or lysosomes). Prior to the appearance of the secondary or specific
granules, it is not morphologically clear which of the granulocytes these promyelocytes will become.

With the appearance of secondary or specific granules at the myelocyte stage, one may now identify the particular
granulocyte lineage. Here, one sees a neutrophilic myelocyte, characterized by a round, flattened or just slightly-indented
nucleus lacking nucleoli. Most notably, an abundance of specific granules is apparent and gives the cytoplasm a
somewhat salmon-colored hue. The primary or azurophilic granules are usually not so apparent at this stage. Following
the myelocyte stage, the nucleus begins to indent more noticeably yielding a neutrophilic metamyelocyte. As the nucleus
continues to thin out, this cell will progress into the neutrophilic band cell, an early stage example of which is seen here.

This image shows two classic neutrophilic band cells, the nuclei of which may begin to fold on themselves, yielding rather
odd-looking nuclei. Continued maturation of the band cell will yield a mature, segmented neutrophil characterized by a
multi-lobed nucleus with the lobes connected by thin strands of chromatin. In this image, note again an
orthochromatophilic erythroblast, and a couple of reticulocytes (compare size and hue to the mature RBCs).
As an example of one of the other granulocytes, compare the morphology of a neutrophilic myelocyte with an eosinophilic
myelocyte. The secondary or specific granules of the eosinophil lineage are noticeably larger than those found in
neutrophils which are barely resolvable and simply give the neutrophil cytoplasm a salmon-colored, slightly grainy texture.

Developing megakaryocytes are impossible to miss given their huge size relative to the other hematopoietic cells. As this
cells develops, its nucleus will become increasingly lobulated as polyploidy develops.
In addition to developing cells corresponding to all the classically-defined developmental stages, appreciate that mitosis is
in integral part of this process. Cells in the midst of mitosis, identified by their condensed chromosomes, are not an
uncommon sight in marrow smears.

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