What is Leukemia?
Leukemia is a blood neoplasm characterized by the proliferation and accumulation of tumor clones in the bone marrow, peripheral blood and lymphoid organs.
Physical examination
The diagnosis is always preceded by the detection of the patient's clinical data ( anamnesis ) and a physical examination, through which the possible presence of swollen lymph nodes or the increase in volume of the liver and spleen is sought. Moreover, the medical examination allows to evaluate: general conditions, fever, sweating, weight loss, infections, previous anemicizations or bleeding episodes.
Blood test
Complete blood count and morphological evaluation by peripheral blood smear are essential for diagnostic orientation.
- Complete blood count
- Cell count: number of red blood cells, leukocytes and platelets.
- Hb level.
- Peripheral blood smear
- The peripheral blood sample, taken from the patient and sent to the analysis laboratory, is subjected to morphological examination under the microscope to ascertain the presence of blasts.
- Determination of blood chemistry parameters : azotemia, glycaemia, transaminases, etc.
- Biochemical profile for renal function, liver enzymes and bilirubinemia, uricemia, LDH, beta-2-microglobulinemia (indicators of kidney and liver function).
In the case of leukemia, the blood test usually shows:
- Anemia : decreased hemoglobin concentration and red blood cell count;
- Thrombocytopenia : decrease in the number of platelets;
- Leukocytosis : increase in the number of leukocytes (less frequently, a condition of leukopenia is observed, with a decrease in the number of white blood cells).
Interpretation of blood test
Reference note: Acute lymphoblastic leukemia = ALL; Acute myeloid leukemia = LMA; Chronic lymphatic leukemia = LLC; Chronic myeloid leukemia = CML.- Most patients show some abnormality in the blood count. The peripheral smear shows the presence of blasts in patients with acute leukemias . In the characterization of the forms of ALL is necessary to resort to the application of immunological techniques for a complete diagnostic definition, unlike the AML, where the morphology and the cytochemistry are sufficiently indicative, to discriminate the different subtypes.
- To diagnose CLL, a variable degree of lymphocytosis must be present (high number of lymphocytes between 10, 000 and 150, 000 / mm3). The absolute neutrophil count is usually normal; the number of red blood cells and platelets has decreased slightly. According to the criteria codified by the FAB group ( French-American-British, which organizes morphological and cytochemical characters in schemes that allow classifying different types of leukemia), a condition to confirm the diagnosis of CLL is represented by the presence of atypical lymphocyte elements (prolymphocytes, immunoblasts and lymphoblasts) less than 10% in the leukocyte formula. Furthermore, it is possible to detect mature lymphocytes with scarce and non-granular cytoplasm, and the presence of shadows of Grumprecht (expression of rupture of trauma cells, a typical finding of CLL).
- CML is defined as white blood cell count: the blood count shows a leukocytosis that can vary from 20 to 300 x 109 / l WBC (WBC = number of white blood cells per liter of blood). The morphological evaluation of peripheral blood reveals mature and immature elements of the neutrophil granulocyte series and an increase in the number of eosinophils, monocytes and / or in particular basophils is often observed. Unlike the leukemic clones of AML, these cells are mature and functional. The number of platelets can be normal (in 60% of cases), increased (30%) or reduced. A picture of modest anemia can be accompanied by findings of leukocytosis and / or thrombocytosis. Leukocyte alkaline phosphatase is generally reduced or absent. Other useful laboratory findings for diagnosis can be represented by the generally high levels of uricemia and serum LDH.
- To classify the AML, appropriate panoptic stains (allowing the simultaneous observation of all blood cells) of peripheral blood and bone marrow smears are used for the morphological characterization. LMA is also diagnosed by demonstrating evidence of particular enzymatic activities and the presence of particular substances believed to be specific for certain cell types (cytochemical characterization).
Bone and spinal cord examination
Bone marrow can be taken in two different ways:
- Bone marrow biopsy
- Bone marrow aspiration
Both procedures, performed under local anesthesia, consist of a puncture of the bone (at the level of the iliac crest, of the sternum or of the femur) to take a small amount of blood from the bone marrow, and a small fragment of bone in the case of biopsy .
The doctor, using the microscope, will examine the sample to try to identify the presence of tumor cells: the bone marrow needle allows a cytological examination to be performed, while the biopsy allows a histological characterization to be performed. The bone marrow sample taken can also be subjected to other diagnostic investigations: morphological examination (microscopic identification of the blasts), cytochemistry, flow cytometry, cytogenetics and molecular biology. Bone marrow aspiration and bone marrow biopsy make it possible to identify the type of leukemia and to define the type of therapeutic strategy to be adopted.
A diagnostic investigation which is sometimes used to further evaluate the evaluation of acute lymphoblastic leukemia and acute myeloid leukemia is rachicentesis, which consists of a lumbar puncture (in the lower back); using a thin needle inserted between the last two vertebrae, a sample of cerebrospinal fluid is taken (liquid that fills the spaces around the brain and spinal cord). The liquor sample will be examined in the laboratory, looking for tumor cells or other signs of alteration.
Interpretative notes on bone marrow examination
- The analysis of a bone marrow sample establishes the diagnosis of leukemia. The morphology of the blasts makes it possible to distinguish between ALL and LMA .
- Bone marrow, in ALL, generally presents with a homogeneous and conspicuous infiltrate by lymphoblasts, small and with poor cytoplasm, which replace the normal elements of bone marrow. For the diagnosis of AML, 30% of the nucleated cells in the aspirate must consist of blasts of myeloid origin.
- Myeloblasts are characterized by the bodies of Auer, which are multiple clusters of granular blue-gray material, which form elongated needles, visible in the cytoplasm of leukemic clones. The presence of Auer bodies is diagnostic for the AML, since these structures do not appear in the ALL.
- In CLL, bone marrow needle aspiration shows lymphocyte infiltration variable between 40% and 95% of total cells.
- In the case of CML, the bone marrow aspirate reveals a marked hypercellularity with hyperplasia of the granulocyte series and often also megakaryocyte. Bone marrow biopsy confirms myeloid hyperplasia with marked reduction of the erythroid compartment and with almost total disappearance of the adipose component. The texture of the reticular fibers of the bone marrow may be normal or slightly increased (marrow fibrosis correlates to the more advanced stages of the neoplasm).
Immunophenotypic analysis
Multiparametric flow cytometry, applied to the cells present in a blood or bone marrow sample, allows a more in-depth characterization of the cell population involved in the pathology: immunophenotyping, following labeling with monoclonal antibodies, allows the identification of specific antigens surface, thus allowing the typing of clones (distinguishes, for example, the monoclonal expansion B or CD5 + in the LLC).
Interpretative notes to the immunophenotypic analysis
- In lymphoid leukemias the determination of the immunophenotype allows the characterization of the lymphocytes: with the cytofluorimetry the origin of the lymphoblasts is identified (distinguishes the B cells from the T). CLL expresses some surface antigens such as CD38, CD19, CD20, CD23, CD52 etc. Moreover, cytometry allows the demonstration of the presence of surface Ig and of the monoclonal expression in lymphoid leukemias (example: all cells express only light chains of the Ig type κ or only type λ). The tumor cells correspond to a minor subpopulation of B cells expressing immunoglobulin M (IgM) and immunoglobulin D (IgD) or the CD5 + antigen associated with the T clones on the cell surface.
- Some specific antigens of the myeloid lineage, such as CD13, CD33, CD41 etc. have been used to diagnose AML : the determination of the immunophenotype through the use of monoclonal antibodies shows more or less specific surface and / or cytoplasmic markers, which allows to identify the different stages of cellular differentiation.
Cytogenetic and molecular analysis
In the laboratory the chromosomes, genes and expression of the transcripts are examined, taken from blood cells, bone marrow or lymph nodes, to establish the type of leukemia.
- Conventional cytogenetic analysis (karyotype reconstruction): investigation that detects the presence of chromosomal abnormalities in pathological cells. This analysis recognizes the "primary" anomalies (present in all abnormal cells), responsible for the early stages of transformation. Identifies the "secondary" alterations responsible for the phases of clonal evolution. It must identify lesions not relevant for the pathogenesis of the disease, as it is a simple expression of genetic instability.
- Molecular cytogenetic analysis : FISH (fluorescent in situ hybridization) is a survey that combines cytogenetic expertise and molecular techniques. The probes marked with fluorochromes allow to detect in the chromosomes or in the interphase nuclei the presence of a DNA sequence of the order of magnitude between tens and hundreds of Kb.
- Molecular biology techniques : PCR (sensitive analytical technique, which detects the presence of "rare" cells), RT-PCR (PCR preceded by reverse transcription) etc.
Interpretative notes on cytogenetic and molecular analysis
- For the diagnosis of Chronic Myeloid Leukemia, cytogenetic tests are indispensable. The Philadelphia chromosome is detectable in 90-95% of CML cases. The use of FISH (fluorescent in situ hybridization) using specific probes for the BCR and ABL genes, allows to quantify the positive Ph clone. The RT-PCR analysis defines the type of BCR / ABL transcript. In particular, the detailed analysis of the three different transcripts (p210, p190, p230), therefore of the different anomalous proteins, has allowed to document that these are more frequently associated to different phenotypes of disease: p210 - frequent in CML, rare in ALL ; p190 - frequent in ALL, rare in CML, rare in AML; p230 - CML with the marked presence of a mature granulocyte population.
- The AML is characterized by numerous chromosomal anomalies that have been and continue to be identified: these allow, in a particular way, to distinguish de novo leukemias (with primitive onset) from secondary ones. The cytogenetic and molecular alterations therefore represent a precise reference to identify specific markers of the different types of AML, which are important for diagnosis and for prognostic implications.
- Cytogenetic analysis of the LLA reveals the presence of clonal chromosomal aberrations in 90% of patients. 30-50% of the forms of ALL have a pseudodiploid karyotype, while 30% have a hyperdiploid structure (alterations in the number of chromosomes). The most frequently encountered structural aberrations are: t (9; 22), t (4; 11), t (8; 14) t (1; 19) t (11; 14) t (7; 14), 6q- .
- The cytogenetic abnormalities found in the CLL include: +12 (trisomy of chromosome 12 present in 25% of cases), 14q +, structural alterations of chromosomes 13, 11, 6, 17 (in particular, deletion of the long arm of chromosomes 13, 6 and 11 and the deletion of the short arm of chromosome 17). Among the biological factors that have occurred we have identified: the mutation of the genes that regulate the production of Ig, the expression of the ZAP-70 protein (tyrosine kinase expressed in normal T lymphocytes: one of its mutations leads to a worse prognosis), the expression of p53 oncogene.
- In ALL, the abnormalities typically found are: the translocation t (8; 21) between chromosomes 8 and 21, which determines the origin of a molecular marker called AML1 / ETO; t (15; 17) and the molecular mutation PML / RAR alpha; alterations involving the 11q23 chromosomal band and chromosome 3.
The doctor, during the formulation of the diagnosis, can prescribe other analyzes, in relation to the manifestation of symptoms and the type of leukemia. These tests could be associated, for example, with a chest x-ray and an ultrasound of the abdomen to highlight a swelling of the lymph nodes or other symptoms, such as an increase in the size of the liver or spleen.
