Acquired Abnormalities (Cancer)

Mutations, including chromosome rearrangements, can affect somatic cells. These mutations are not transmissible to the offspring of the individual, but can affect successive generations of cells. These are the acquired (chromosome) abnormalities, or tumours. If all, or many different cases of a tumour show one consistent chromosome abnormality, the abnormality could be involved in the causation (etiology) of the disease, perhaps because the abnormality has altered the normal function of a gene or genes required for the normal control of the cell cycle. Here we will concentrate on neoplasias of blood cells (leukaemias), as these are relatively easier to study when compared with cells from solid tumours.

Before considering the pathology associated with acquired chromosome abnormalities in blood neoplasias, it may be useful to be aware of three points; the morphologic and functional diversity of cell types found in the blood and marrow, the process of their formation, or haemopoiesis, and the factors involved in leukaemia classification.

1. The morphology of the different cellular types that are present in normal blood, their nomenclature and standard range values found in normal individuals are summarised below. For those interested in the morphology of blood cells, a Leishman-stained blood smear can be studied at this site from the University of Western Australia. There is also a section on normal blood in the Haematology Malignancy Diagnostic Service (HMDS) from Cookridge Hospital & Leeds General Infirmary, although this site is much larger (see below).

Morphology		Function	Normal range
White cells; (1) Granulocytes come in three types, neutrophils, eosinophils, basophils.		Granules are lysosomal.	x 10^9 per l.
		Nucleus stained purple and divided into 2/5 lobes united by thin strands. In women some neutrophils show a tiny 'drumstick'. Produced in the marrow, move to sites of infection or inflamation to phagocyte. Chemotaxis determined by cellular signals.	The most common nucleated cell seen in blood film. 1.7-6.1 (M) 1.7-7.5 (F) Left shift means less differentiated forms (nuclei less labulated), right shift the opposite.
		Similar to neutrophils but cytoplasmic granules are coarser and redder. Rarely more than three nuclear lobes. Role in allergic response, defence against parasites and removing fibrin formed during inflamation.	0.04-0.4
		Uncommon in perypheral blood. Many cytoplasmic dark granules containing heparin and histamine.	0.01-0.1
White cells (2); Mononuclear cells come in two types; Monocytes and lymphocytes.
		Larger than other blood leukocytes, nucleus large oval or simply indented. Blue abundant cytoplasm. They become macrophages in the tissues, where they phagocyte. Secrete gowth factors (IL6,TNF,IL1, IL3...)	0.2-0.8
		Round solid nucleus, little cytoplasm. B-cells and plasma cells produce immunoglobulins, divided into 5 subtypes; IgA, M, D, E and G. IgM is produced first in response to antigen, then IgG (see below) T-lymphocytes are 'helper' or 'killer' cells, and produce TCR (See below)	1.5-3.5 80% are T-cells, 20% are B-cells.
		Clot formation
Red cells (erytrhocytes, seen above)		Carry oxygen

Cell function. The immunoglobulin chain and the T-cell receptor chain- B-lymphocytes produce immunoglobulins (antibodies, see below), and are involved in tagging invaders for phagocytosis, complement activation and killer T cell activation. T-lymphocytes produce T-cell receptor, and can be 'helper' cells, presenting antigens to B-lymphocytes, or 'killer' cells, killing target cells by secretting perphorin. Here we should mention some basic details of their structure as this relates to the cytogenetic observations made on lymphocytic leukaemias.

Molecule	Chain	Gene locus
Immunoglobulin (basic structure).
	Heavy, V,D,J & C, each gamma, mu, alpha,delta or epsilon	14q32
	Light kappa, V,D,J,C,	2p12
	Light lambda, V,D,J,C,	22q11
Immunoglobulin types
	IgA	Secreted form
	IgD, IgG,IgE	G is the most common form
	IgM	First response to antigen
T-cell receptor	Chain	Gene locus
	Alpha & Delta	14q11
	Beta	7q34
	Gamma	7p15

The table above is of interets to cytogeneticists because it makes some sense of the observation that in B-cell leukaemias the breakpoints involved areoften in the bands containing the genes for immunoglobulins, while in T cell leukaemias the most common breakpoints involve the genes for the T-cell receptor chains.

2. Haemopoiesis.- All the cell types described above originate from bone marrow stem cells, in the process of haemopoiesis. An understanding of the pathology of blood cell formation requires some understanding of the normal process, and this is summarised in the figure below. In brief, you see that there is in the marrow a reservoir of 'stem cells', very undifferentiated. These cells divide asymetrically; one of the daughter cells remains a stem cell, the other makes progress in the process of differentiation, along three possible lineages; a lymphoid lineage (eventually producing B ot T lymphocytes or plasma cells), a myeloid lineage (producing either granulocytes, or monocytes) and an erythroid lineage (ending as eryhtrocytes or platelets).You could learn more on normal haemopoisesis by visiting Marylin Pike's website, from where this diagram was taken;

To identify the progenitors of each cell type the criteria from morphology often need to be supplemented with details of cytochemistry or protein markers on the cell surface, nucleus or cytoplasm, (immunophenotype), which characterise different cellular types and their differentiation stages. A consensus paper on the immunophenotyping characteristics of normal and leukaemic cells can be seen at the website from the European Working Group on Clinical Cell Analysis (EWGCCA).

3.- Leukaemia definition and classification.- The Leukaemias may be characterised as malignancies of primitive haemopoietic cells, resulting in disturbed proliferation, differentiation or cell death. If the abnormal cell is a myeloid precursor, the leukaemia is called 'myeloid'. If a lymphoid stem cell is involved, the leukameia is called 'lymphoid'. If the abnormal primitive cell can proliferate but not differentiate, the leukameia is called 'acute', and an accumulation of undifferentiated or 'blast' cells is observed. If the primitive cell retains the ability to differentiate, the cells that accumulate (mainly mature cells) may do so because the normal process of apoptosis or self-destruction is impaired. These are termed 'chronic leukaemias'. Notice that 'acute' or 'chronic' when speaking of leukaemias refer only to the morphology of the leukaemia, and are not an indication of the time course or severity of the disease. From the above you have four types of Leukaemia;

• Acute Myeloid, (AML)
• Acute Lymphoid, (ALL)
• Chronic Myeloid and (CML)
• Chronic Lymphoid (CLL)

The French American British (FAB) system of sub-classification of leukaemias takes into account data from the clinic, the blood and marrow morphology, immunophenotype and cytogenetics.The system works best for the myeloid leukaemias. The result is that each of the four types mentioned above can be subdivided into different and more homogeneous clinical entities. Cytogenetic results help to define some of these subtypes, and in some cases are good predictors of patient's reaction to treatment (prognosis).

The FAB classification of lymphoid leukaemias is not very satisfactory and it has been revised in the Revised European American Lymphoma classification (REAL). The criteria for this subclassification derive from the nature and degree of differentiation of the tumour cell involved in each case, as defined by morphology and expression of specific cell markers (such as the stage in the process of immunoglobulin production). A summary of the REAL system of classification as applied to lymphoid leukaemias can be seen at the HMDS site.

Chromosomal rearrangements that are characteristic of a disease point to the location of genes possibly involved in its etiology, genes which, when de-regulated because of their new cytogenetic position, alter normal processes such as apoptosis or the course of the cell cycle. Once again, the pathology section of the HMDS site integrates all these observations. The following is a summary of frequent cytogenetic abnormalities found in haematologic neoplasias. Further down in this page you can explore some extensive databases that are useful to the clinical cytogeneticist dealing with tumours, mostly haematologic.

Rearrangement	Chromosomes	Genes	Disease	Prognosis
t(1;19)		E2A PBX	Pre B ALL
t(1;7)			MDS
t(2;8)			ALL (variant of Burkitt's type)
t(4;11)		MLL AF4	Pre pre B ALL
del5(q)			MDS
del7(q)			MDS
t(8;14)			ALL (Burkitt's)
t(8;21)			AML-M2
t(8;22)			Variant ALL (Burkitt's)
t(9;22)(q34;q11)		ABL(9) BCR(22)	CML AML ALL
t(14;18)
t(15;17)			AML-M3
t(16:16)			AML-M4
i(17q)			CML AML ALL

There are many other chromosomal abnormalities, numerical as well as structural, that are found in the leukaemias. To read more about them you are addressed to any of the specialist databases below.

• Atlas of Genetics and Cytogenetics in Cancer. From the University of Wisconsin. This is the smallest of the three databases and shows well organised partial karyotypes of the most common abnormalities associated with cancer, specially leukaemia. If you are a cytogeneticist, your training should include discovering as many of these chromosome abnormalities as possible.
• Recurrent Chromosome Aberrations in Cancer. Felix Mitelman's classic catalogue on line. Very large and informative, but you want to know what you are looking for.
• Atlas of Genetics and Cytogenetics in Oncology and Haematology. By Jean-Loup Huret in Poitiers. Very clear, multidisciplinary and well cross-referenced.
• Medical students can access Takuji Ichihasi's Atlas of Haematology from the University of Nagoya to view blood and marrow smears from haematologic malignancies stained in different ways.

Exercise 4. You have found translocation t(15;17)(q22;q21) in a bone marrow sample from a young (20 years old) male showing disseminated intravascular coagulation. Explore one or more of the databases above to gain some information about the likely diagnosis. (However, keep in mind that the diagnosis is always made by the clinician who sees the patient).

Compare the above situation with the finding of translocation t(4;11)(q21;q23) in a two year old girl with Acute Lymphoblastic Leukaemia.