Initial Diagnostic Work-Up of Acute Leukemia

The treating clinician should provide relevant clinical data or ensure that these are readily accessible by the pathologist. Note: These data include, but are not limited to, the patient’s age, sex, and ethnicity; history of any hematologic disorder or known predisposing conditions or syndromes; any prior malignancy; exposure to cytotoxic therapy, immunotherapy, radiotherapy, or other possibly toxic substances; and any additional clinical findings of diagnostic or prognostic importance. The treating clinician should also include any history of possibly confounding factors, such as recent growth factor therapy, transfusions, or other medications that might obscure or mimic the features of AL. The treating clinician should also obtain and provide information regarding any family history of any hematologic disorders or other malignancies.

The treating clinician should provide relevant physical examination and imaging findings or ensure that those results are readily accessible by the pathologist. Note: This includes, but is not limited to, neurologic examination findings and the presence of tumor masses (e.g. mediastinal), other tissue lesions (e.g. cutaneous), and/or organomegaly.

The pathologist should review recent or concurrent complete blood counts and leukocyte differentials and evaluate a peripheral blood smear.

The treating clinician or pathologist should obtain a fresh bone marrow aspirate for all patients suspected of AL, a portion of which should be used to make bone marrow aspirate smears for morphologic evaluation. The pathologist should evaluate an adequate bone marrow trephine core biopsy, bone marrow trephine touch preparations, and/or marrow clots, if available, in conjunction with the bone marrow aspirates. Note: If bone marrow aspirate material is inadequate or if there is a compelling clinical reason to avoid bone marrow examination, peripheral blood may be used for diagnosis and ancillary studies if sufficient numbers of blasts are present. If a bone marrow aspirate is unobtainable, touch imprint preparations of a core biopsy should be prepared and evaluated, and an additional core biopsy may be submitted, unfixed in tissue culture medium, for disaggregation for flow and genetic studies. Optimally, the same physician should interpret the bone marrow aspirate smears and the core biopsy specimens, or the interpretations of those specimens should be correlated if performed by different physicians.

In addition to performing morphologic assessment (blood and bone marrow), the pathologist or treating clinician should obtain sufficient samples and perform conventional cytogenetic analysis (i.e., karyotype), appropriate molecular genetic and/or FISH testing, and FCI. The flow cytometry panel should be sufficient to distinguish AML (including APL), including early T-ALL, B-ALL, and AL of ambiguous lineage in all patients diagnosed with AL. Molecular genetic and/or FISH testing does not, however, replace conventional cytogenetic analysis. Note: If sufficient blood marrow aspirate or peripheral blood material is not available for FCI, immunohistochemical studies may be used as an alternative method for performing limited immunophenotyping. In addition, a second bone marrow core biopsy can be obtained and submitted, unfixed in tissue culture medium, for disaggregation for genetic studies and flow cytometry.

For patients with suspected or confirmed AL, the pathologist may request and evaluate cytochemical studies to assist in the diagnosis and classification of AML.

The treating clinician or pathologist may use cryopreserved cells or nucleic acid, formalin-fixed, nondecalcified paraffin-embedded tissue, or unstained marrow aspirate or peripheral blood smears obtained and prepared from peripheral blood, bone marrow aspirate, or other involved tissues for molecular or genetic studies in which the use of such material has been validated. Such specimens must be properly identified and stored under appropriate conditions in a laboratory that is in compliance with regulatory and/or accreditation requirements.

For patients with ALL receiving intrathecal therapy, the treating clinician should obtain a CSF sample. The treating clinician or pathologist should ensure that a cell count is performed and that examination and enumeration of blasts on a cytocentrifuge preparation are performed and are reviewed by the pathologist.

For patients with AL other than those with ALL who are receiving intrathecal therapy, the treating clinician may, under certain circumstances, obtain a CSF sample when there is no clinical contraindication. The treating clinician or pathologist should ensure that a cell count is performed and that examination and enumeration of blasts on a cytocentrifuge preparation are performed and are reviewed by the pathologist.

For patients with suspected or confirmed AL, the pathologist may use flow cytometry in the evaluation of CSF.

For patients who present with extramedullary disease without bone marrow or blood involvement, the pathologist should evaluate a tissue biopsy specimen and process it for morphologic, immunophenotypic, cytogenetic, and molecular genetic studies, as recommended for the bone marrow. Note: Additional biopsies may be indicated to obtain fresh material for ancillary testing.

For patients with suspected or confirmed AL, the pathologist or treating clinician should ensure that flow cytometry analysis or molecular characterization is comprehensive enough to allow subsequent detection of MRD.

For pediatric patients with suspected or confirmed B-ALL, the pathologist or treating clinician should ensure that testing for t(12;21)(p13.2;q22.1); ETV6-RUNX1, t(9;22)(q34.1;q11.2); BCR-ABL1, KMT2A (MLL) translocation, iAMP21, and trisomy 4 and 10 is performed.

For adult patients with suspected or confirmed B-ALL, the pathologist or treating clinician should ensure that testing for t(9;22)(q34.1;q11.2); BCR-ABL1 is performed.

In addition, testing for KMT2A (MLL) translocations may be performed.

For patients with suspected or confirmed ALL, the pathologist or treating clinician may order appropriate mutational analysis for selected genes that influence diagnosis, prognosis, and/or therapeutic management, which include, but are not limited to, PAX5, JAK1, JAK2, and/or IKZF1 for B-ALL and NOTCH1 and/or FBXW7 for T-ALL. Testing for overexpression of CRLF2 may also be performed for B-ALL.

For pediatric and adult patients with suspected or confirmed AML of any type, the pathologist or treating clinician should ensure that testing for FLT3-ITD is performed.

The pathologist or treating clinician may order mutational analysis that includes, but is not limited to, IDH1, IDH2, TET2, WT1, DNMT3A, and/or TP53 for prognostic and/or therapeutic purposes.

For adult patients with confirmed CBF AML (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22); CBFB-MYH11), the pathologist or treating clinician should ensure that appropriate mutational analysis for KIT is performed.

For pediatric patients with confirmed CBF AML; RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22); CBFB-MYH11, the pathologist or treating clinician may ensure that appropriate mutational analysis for KIT is performed.

For patients with suspected APL, the pathologist or treating physician should also ensure that rapid detection of PML-RARA is performed. The treating physician should also order appropriate coagulation studies to evaluate for disseminated intravascular coagulation.

For patients other than those with confirmed CBF AML, APL, or AML with myelodysplasia-related cytogenetic abnormalities, the pathologist or treating clinician should ensure that mutational analysis for NPM1, CEBPA, and RUNX1 is also performed.

For patients with confirmed AL, no recommendation is made for or against the use of global or gene-specific methylation, microRNA expression, or gene expression analysis for diagnosis or prognosis.

For patients with confirmed mixed-phenotype AL, the pathologist or treating clinician should ensure that testing for t(9;22)(q34.1;q11.2); BCR-ABL1, and KMT2A (MLL) translocations is performed.

All laboratory testing performed for the initial work-up and diagnosis of a patient with AL must be performed in a laboratory that is in compliance with regulatory and/or accreditation requirements.

If, after examination of a peripheral blood smear, it is determined that the patient will require immediate referral to another institution with expertise in the management of AL for treatment, the initial institution should, whenever possible, defer invasive procedures, including blood marrow aspiration and biopsies, to the treatment center to avoid duplicate procedures, associated patient discomfort, and additional costs.

If a patient is referred to another institution for treatment, the primary institution should provide the treatment center with all laboratory results, pathology slides, flow cytometry data, cytogenetic information, and a list of pending tests at the time of the referral. Pending test results should be forwarded when they become available.

In the initial report, the pathologist should include laboratory, morphologic, immunophenotypic, and, if performed, cytochemical data, on which the diagnosis is based, along with a list of any pending tests. The pathologist should issue addenda/amended reports when the results of additional tests become available.

The pathologist and treating clinician should coordinate and ensure that all tests performed for classification, management, predicting prognosis, and disease monitoring are entered into the patient’s medical records.

Treating physicians and pathologists should use the current WHO terminology for the final diagnosis and classification of AL.