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Diagnosing Acute Lymphoblastic Leukemia


Medical editor Lorentz Brinch MD
Hematologist
Oslo University Hospital

General

Acute lymphoblastic leukemia (ALL) often grows rapidly, and quickly accomplished examinations with treatment starting after 1-2 days may be significant to save a patient with a large tumor mass.

For the choice of treatment, it is critical to have a comprehensive morphological, immunological, and cytogenetic characterization of the tumor cells.

ALL is separated into 3 subgroups (L1-L3). The grouping is based on the morphological examination after the FAB classification of blood and bone marrow smears and is supported by immunophenotyping, cytogenetic, and molecular genetic examinations as well as combinations of these techniques. WHO staging is increasingly applied also in Norway. 

ALL and lymphoblastic lymphoma have many similarities. Both diseases originate from rapid proliferating cells with blast morphology. Conditions with more than 25% lymphoblasts in bone marrow are considered acute lymphoblastic leukemia. If the bone marrow criteria is met, the condition is leukemia even with mediastinal tumors or other lymph node tumor.

Burkitt's lymphoma/leukemia (L3) originates from more mature B lymphocytes and requires special treatment. The treatment is determined by the type of tumor cells and not whether the disease manifests itself as leukemia or lymphoma. 

Indication

  • Suspicion of acute lymphoblastic leukemia

Goal

  • Confirm the diagnosis 
  • Identify risk group for disease progression
  • Cure the disease

 

 

 

 

 

 

 

 

Norsk selskap for hemtaologi. Handlingsprogram for diagnostikk og behandling av akutt lymfoblastisk leukemi/lymfoblastisk lymfom og burkitt lymfom leukemi hos voksne [Online]. Mars 2006 [hentet 15. april 2007]; tilgjengelig fra: URL:http://www.legeforeningen.no/asset/33493/1/33493_1.doc


Definitions

May-Grünwald/Giemsa staining

In order to differentiate between cell structures, the May-Grünwald/Giemsa stain is used (MGG stain). This staining method works by a chemical reaction between stain substances and the biochemical components of the cell. The stain substances are neutral salts which ionize in water and react with ions in the cell. MGG staining is the basis for the FAB classification. 

There are no other special examinations as useful as microscopy of MGG-stained blood and bone marrow smears to differentiate between different types of leukemia.

Flow cytometry immunophenotyping

Flow cytometry immunophenotyping is necessary to clarify the origin of the leukemic clone and for adequate characterization of leukemic cells included for later use for MRD diagnostics.

Cytogenetic and molecular genetic examinations

Using genetic technology methods, for example FISH analysis, (fluorescent in situ hybridization) or PCR (polymerase chain reactions), details are revealed of gene material which change places, lacks or changes of chromosomes, and the significance this has for differentiation and growth of leukemia cells. This has an increasing significance for treatment since the methods are very sensitive for the presence of characteristic gene material from leukemia cells. The most important example of this is evidence of BCR-ABL. PCR will identify BCR-ABL regardless of where the gene is located in the genome, and is today the most sensitive method for finding the fusion gene.

The presence of this fusion gene indicates a poor prognosis using normal chemotherapy. The Philadelphia chromosome (which is a shortened chromosome 22) occurs due to a translocation between chromosome 9 and 22, where the fusion gene BCR-ABL is created .


Examinations

Bone marrow aspiration

A bone marrow aspiration or bone marrow biopsy if it is difficult to aspirate representative bone marrow, is necessary to diagnose ALL. A biopsy can in some cases also provide additional valuable information.

Examinations performed on the aspirate:

  • Microscopy, MGG staining, and possible cytochemical staining
  • Immunophenotyping (flow cytometry)
  • Cytogenetics with directed testing using FISH or molecular genetic techniques
  • If possible, a cell suspension which is frozen locally for later supplementary examinations.
  • RNA/DNA extraction for later molecular genetic analyses should be done, if possible.

In all patients, the bone marrow aspirate and blood are sent to PCR for BCR-ABL.

Blood tests

General:

ABO typing, hemoglobin, leukocytes with differential counting, thrombocytes, blood smear, LD, albumin, creatinine, Na, K, Ca, phosphate, urea, glucose, CRP, CMV.

Special testing:

  • Blood tests at the start of treatment with emphasis on development of tumor lysis syndrome: urate, Ca, Mg, K+, phosphate, creatinine, and liver tests.  
  • HLA typing – taken if there is a need for HLA compatible blood components

Lumbar puncture

A lumbar puncture is performed when there is a clinical suspicion of CNS leukemia or for the first intraspinal prophylaxis treatment. 

Examinations performed on the aspirate:

  • Cell count - for an increase in cell count, cytospin with MGG staining is performed
  • Spinal protein

In some cases, a cytological examination of the spinal fluid is done and possibly with immunophenotyping.

Interpretations of findings in spinal fluid may be difficult when there are blasts in blood due to the risk of contamination by puncture bleeding.

Familial evaluation

In cases where an allogeneic stem cell transplant is considered, HLA typing of siblings and parents is done when remission is achieved. This means all patients under 60 who reach complete remission.

 


Findings

Acute lymphoblastic leukemia is divided into early B-cell population (about 80%), T-cell (about 10-15%), and B-cell with surface immunoglobulin (< 5 %).

Patients with Philadelphia chromosome-positive acute lymphoblastic leukemia, have a poorer prognosis, and represent more than 20% of adult cases.

About 95% of all types of acute lymphoblastic leukemia (except for B-cell) have elevated terminal deoxylnucleotidyl transferase (TdT) . If this marker is negative, the diagnosis of acute lymphoblastic leukemia is doubtful.

Acute B-lymphoblastic leukemia / B-lymphoblastic lymphoma, L1  or L2

  • Originates from precursor B-cells
  • Defined by lymphoblastic morphology L1 or L2
    • L1 – blasts with very little cytoplasm (< 15 of nucleus area), and less than 5% promyelocytes, negative diaminobenzidine peroxidase (DAB staining)
    • L2 – less than 5% promyelocytes, more cytoplasm than L1
  • Immunophenotype like immature B-cells

Acute T-lymphoblastic leukemia / T-lymphoblastic lymphoma, L1  or L2

  • Originates from precursor T-cells 
  • Defined by lymphoblastic morphology L1 or L2
    • L1 – blasts with very little cytoplasm (< 15 of nucleas area), and less than 5% promyelocytes, negative diaminobenzidine peroxidase (DAB staining).  
    • L2 – less than 5% promyelocytes, more cytoplasm than L1
  • Immunophenotype like immature T cells
  • CD1a is negative for Pro tT and mature T-ALL, positive for cortical T-ALL

Burkitt's leukemia/ lymphoma L3

  • Orginates from more "mature" B-cells 
  • Defined by lymphoblastic morphology (deep basophilic cytoplasm, vacuoles, FAB L3)
  • Immunophenotype like mature B-cells
  • Characteristic karyotype
  • These leukemia cells usually have three translocations: t(8;14), t(2;8), and t(8;22).

Follow-up

As soon as the diagnosis is made, treatment is initiated.

Prognostic factors

High-risk for acute B-lymphoblastic leukemia:

  • Philadelphia chromosome/bcr-abl/t(9;22)(q34;q11)
  • t(4;11)(q21;q23) and other 11q23 anomalies
  • massively hypodiploid karyotype
  • complex karyotype, that is > 5 discrepancy
  • leukocytes in blood > 30 x 109/l
  • ≥ 5 % blasts in bone marrow smear 4 weeks after starting treatment
  • positive MRD-status 12 months after time of diagnosis

High-risk for T-cell illness:

  • ≥ 5 % blasts in bone marrow smear 4 weeks after starting treatment
  • pro T-ALL
  • mature T-ALL
  • positive MRD status 12 months after time of diagnosis

All others are considered standard risk.

Minimal residual disease (MRD)

In younger patients (< 60), it is recommended to try to define the leukemia-specific marker with immunophenotyping or molecular genetic method at the time of diagnosis and follow the MRD level during treatment when the clone-specific marker can be defined. If, as in the case of high-risk leukemia, a decision to use allogeneic stem cell transplant as primary consolidation treatment has been made, or this is clearly inappropriate (age > 60), the MRD determination has no practical consequences and can be left out.

It is recommended that the MRD is determined in the bone marrow aspirate at the concluded consolidation treatment (before starting maintenance treatment) and 12 months after the time of diagnosis, if clone-specific markers are successfully defined at the time of the diagnosis. Examinations should be avoided in the aplasia phase since the chance for an unsuccessful test is the greatest.

In order for the MRD analysis to be emphasized, the sensitivity of the analysis must be at least 10-4. Ideally, the analysis should be carried out on two clone-specific markers. If the results are difficult to interpret, the analysis should be repeated.

  • With positive MRD-status (> 10–4) after concluded induction (about week 16) an allogeneic stem cell transplantation should be considered. Alternatively, the MRD level is followed with a new test after 1 month. Increasing MRD (1 log unit or more) indicates a high risk for recurrence.
  • With positive MRD-status (> 10–4) after 12 months, an allogeneic stem cell transplantation is considered. For transition from previous negative to positive status which is not due to variation in test sensitivity, supplementary reinduction treatment is considered. Contact the Section for Blood Diseases for planning further treatment.

It is currently recommended that all patients complete full maintenance treatment even if MRD is negative in week 16 and 12 months. Rapid decline of MRD (negative in week 2 and week 16) indicates good prognosis.

Knowledge of the use of MRD is under rapid development.


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