Acute Leukemia Testing Guidelines

· CCO Dec. 2016 Consensus Pathology Recommendations for Complex Malignant Hematology

· Testing Methodologies:

· Biomarkers can be assessed by a variety of techniques including, but not limited to, immunohistochemistry (IHC), in-situ hybridization (ISH and FISH), Gband karyotyping, array comparative genomic hybridization (aCHG) and a variety of other techniques including those using polymerase chain reaction (PCR) and/or nucleic acid sequencing.

· Technologies to identify biomarkers continue to change and laboratory infrastructure can be variable, making it important that each laboratory determine which technologies and laboratory processes are best suited for assessment of each biomarker to meet clinical need in a cost-effective manner. It is the responsibility of the laboratory in conjunction with established Communities of Practice to ensure that minimum established biomarkers and parameters of test performance are met, regardless of the methods or platforms chosen.

· Panel testing will increase efficiencies in testing and allow standardization of biomarker testing within the province.

· AML:

· Tests to Perform (required):

· The list of useful genomic biomarkers for management of AML is large and continues to evolve. It should be re-visited on an annual basis.

· A Community of Practice should determine an appropriate list of essential biomarkers for Ontario patients with myeloid neoplasms. Panel testing will increase efficiencies in testing and allow standardization of biomarker testing within the province.

· Cytogenetics / karyotyping (G-band / FISH analysis)

· Evaluation of genomic biomarkers for any or all of diagnostic sub-classification, prognosis and therapeutic guidance (Appendix B – Table 5)

· ASXL1

· BCOR

· BRAF

· CALR

· CEBPA*

· DNMT3A

· EZH2

· FLT3 (ITD)*

· IDH1

· IDH2

· JAK2

· KIT

· KMT2A (PTD)

· MPL

· NPM1*

· NRAS

· RAD21

· RUNX1

· SF3B1

· SRSF2

· STAG2

· TET2

· TP53

· U2AF1

· WT1

· ZRSR2

· (* Analysis required within 5 days or less)

· Preparation for HLA Typing when appropriate

· MRD testing and quantitative monitoring:

· (PML/RARA, RUNX1/RUNX1T1, CBFB/MYH11, BCR-ABL1, NPM1) or other selected cytogenetic alterations

· Schedule:

· At diagnosis

· At 6 weeks

· q 3 months for years 1 & 2

· q 6 months for year 3

· at year 4

· mRNA expression analysis and miRNA analysis is not required, but there is emerging evidence that these markers may be useful for prognosis and management of AML in the future (Appendix B – Table 6)

· BAALC

· DNMT3B

· ERG

· MECOM/EVI1

· microRNA (miR-155 / 181a / 3151)

· MN1

· SPARC

· TATs (calendar days):

· Initial diagnosis of AML - (within 48 hours)

· PML/RARA - (within 24 hours)

· BCR-ABL1 - (within 5 days)

· FLT3(ITD), CEBPA, NPM1 – (within 5 days)

· G-band karyotype / FISH analysis – (within 14 days)

· Complete genomic characterization – (within 21 days)

· HLA Typing - (within 14 days)

· ALL:

· Tests to Perform (required):

· The list of useful genomic biomarkers for management of ALL is large and continues to evolve. It should be re-visited on an annual basis.

· A Community of Practice should determine an appropriate list of essential biomarkers for Ontario patients with ALL.

· Cytogenetics/ karyotyping (G-banding/ FISH Analysis)

· Evaluation of genomic biomarkers for diagnostic sub-classification per current WHO diagnostic categories (Appendix A – Table 2)

· Preparation for HLA Typing when appropriate

· B-cell and T-cell gene rearrangements (when appropriate)

· MRD testing:

· Analysis of tumor/patient-specific immunoglobulin or T-cell receptor gene rearrangements

· BCR-ABL1 monitoring every 3 months (in BCR-ABL1 positive cases).

· Monitoring frequency for other genes continues to evolve and will require further investigation.

· Other biomarkers and evaluation methods for MRD testing in ALL may need to be established

·

· TATs (calendar days):

· Diagnosis of ALL (without most genomic biomarkers for sub-classification) – (within 48 hours)

· Analysis of BCR-ABL1 – (within 5 days).

· Cytogenetic/G-band karyotype, FISH analysis – (within 14 days)

· Tumor-specific immunoglobulin or T-cell receptor gene rearrangements for clonotypic analysis – (within 14 days when required for diagnosis; within 28 days when required for MRD testing alone)

· HLA Typing – (within 14 days)

· MDS:

· Tests to Perform (required):

· The list of useful genomic biomarkers for management of MDS is large and continues to evolve. It should be re-visited on an annual basis.

· Community of practice should determine an appropriate list of essential biomarkers for Ontario patients with MDS.

· Cytogenetics (G-banding karyotype)

· Evaluation of additional genomic biomarkers as required for any or all of diagnostic sub-classification, prognosis and therapeutic guidance (Appendix A – Table 3 and Appendix B – Table 5)

· Preparation for HLA Typing when appropriate

· TATs:

· Initial diagnosis of MDS (if it can be made without genomic testing) – (within 48 hours)

· Cytogenetic/G-banding karyotype, FISH – (within 14 days)

· Complete genomic characterization – (within 21 days)

· Aplastic Anemia:

· The suggested minimal diagnostic workup for patients with aplastic anemia is outlined in Appendix D – Table 8

· Peripheral blood chromosomal breakage analysis: diepoxybutane test (DEB Test) for possible FA if patient aged < 50 years, or clinically suspected.

· Emerging diagnostic tests: the following are not currently routine diagnostic tests, but are likely to be so within the next few years

· Peripheral blood leucocyte telomere length:

· Useful for disease screening for telomere gene mutations in classic DC; less specific in adult onset AA with TERC/TERT mutations; short telomeres may also occur in acquired AA with reduced stem cell reserve (Townsley et al, 2014)

· Next generation sequencing, gene panels for:

· Telomere gene complex mutations

· Other IBMFS

· Acquired somatic mutations, typical of myeloid malignancies, to help distinguish AA from hypocellular MDS and for early detection of clonal evolution to MDS/AML (Kulasekararaj et al, 2014)

· SNP array karyotyping (microarray)

· Whole genome scanning to detect unbalanced chromosomal defects (Afable et al, 2011a)

· Routine extensive biomarker testing is not indicated for Aplastic Anemia at this time, unless suspicion arises for the development of a myeloid neoplasm.

· Patients with Aplastic Anemia need to have MDS / AML excluded and be monitored for the development of MDS / AML, and PNH.

· Patients considered for stem cell transplant should have HLA testing performed as soon as transplant is being considered.

· Aplastic Anemia patients may develop myeloid neoplasm-associated mutations seen in MDS and AML. Some biomarkers have increased frequency compared to MDS/AML include PIGA, BCOR, and BCORL1 (Yoshizato et al, 2015); patients with suspected myeloid neoplasms should be investigated as outlined for AML/MDS.

· CAP ASH Guidelines:

· Specimen Requirements and Tests to Request:

· Core biopsy adequacy:

· In general, > 1 cm or larger is considered optimal

· Must contain intact hematopoietic regions of the BM

· Cannot consist largely of cortical or subcortical regions

· Touch preparations should be made for every core biopsy (unstained initially)

· Clot specimen should be made from the remaining BM aspirate specimen, once adequate numbers of BM aspirate specimen slides have been prepared

· The treating clinician should provide relevant clinical data or ensure that this is readily accessible by the pathologist. (Strong recommendation) 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 acute leukemia. The treating clinician should also obtain and provide information regarding any family history of any hematologic disorders or other malignancies

· The use of recombinant granulocytic growth factors, such as granulocyte colony-stimulating factor and granulocytemacrophage colony-stimulating factor, may transiently increase blasts in the blood and/or BM, which, in some cases, may account for 20% or more of the cells and lead to an erroneous diagnosis of AML. The increase in blasts may persist up to 5 weeks after cessation of growth factor therapy.

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

· The pathologist should review recent or concurrent complete blood cell (CBC) 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 acute leukemia, a portion of which, should be used to make bone marrow aspirate smears for morphologic evaluation. If performed, the pathologist should evaluate an adequate bone marrow trephine core biopsy, bone marrow trephine touch preparations, and/or marrow clots, in conjunction with the bone marrow aspirates. (Strong recommendation) Note.—If bone marrow aspirate material is inadequate or if there is 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 morphologic assessment (blood and bone marrow), the pathologist or treating clinician should obtain sufficient samples and perform conventional cytogenetic analysis (ie, karyotype), appropriate molecular genetic and/or fluorescent in situ hybridization (FISH) testing, and flow cytometric immunophenotyping (FCI).

· Molecular genetic and/or FISH testing should be considered complementary to an adequate conventional cytogenetic analysis.

· The flow cytometry panel should be sufficient to distinguish acute myeloid leukemia (including acute promyelocytic leukemia), T-cell acute lymphoblastic leukemia (T-ALL) (including early T-cell precursor leukemias), B-cell precursor ALL (B-ALL), and acute leukemia of ambiguous lineage on all patients diagnosed with acute leukemia. Molecular genetic and/or FISH testing does not, however, replace conventional cytogenetic analysis. Strong recommendation Note.—If sufficient bone 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 media, for disaggregation for genetic studies and flow cytometry.

· Although no standard FCI panels are mandated for all laboratories, there are recommendations for instrumentation, preanalytic variables, panel design, data analysis, and validation by the EuroFlow Consortium (Leiden, the Netherlands),149 the British Committee for Standards in Haematology (London, United Kingdom),150 and the International Clinical Cytometry Society (Glenview, Illinois).

· Sample type for molecular or genetic studies:

· The treating clinician or pathologist may use cryopreserved cells or nucleic acid, formalin fixed, nondecalcified paraffin-embedded (FFPE) 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.

· In general, the use of FFPE cells has been most successful for DNA-based analyses, whereas RNA extracted from such specimens is fragmented by formalin fixation and is often of poor quality. Nevertheless, the expression pattern of small RNAs, eg, miRNAs, extracted from FFPE is reportedly similar to that derived from cryopreserved cells.

· For patients with acute lymphoblastic leukemia (ALL) receiving intrathecal therapy, the treating clinician should obtain a cerebrospinal fluid (CSF) sample. The treating clinician or pathologist should ensure that a cell count is performed and that examination/enumeration of blasts on a cytocentrifuge preparation is performed and is reviewed by the pathologist.

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

· 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 acute leukemia for treatment, the initial institution should, whenever possible, defer invasive procedures, including bone marrow aspiration and biopsies, to the treatment center to avoid duplicate procedures, associated patient discomfort, and additional costs. Strong recommendation

· 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. Strong recommendation

·

· Tests to Perform in the Lab:

· For patients with suspected or confirmed acute leukemia, the pathologist or treating clinician should ensure that flow cytometry analysis or molecular characterization is comprehensive enough to allow subsequent detection of minimal residual disease (MRD).

· ALL:

· 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); BCRABL1, KMT2A (MLL) translocation, iAMP21, and trisomy 4 and 10 is performed

· Certain abnormalities encountered in ALL, such as t(12;21)(p13.2;q22.1) ETV6-RUNX1 fusion or intrachromosomal amplification of chromosome 21 (iAMP21), can be cytogenetically cryptic and are optimally detected by interphase or metaphase FISH.

· 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. Strong recommendation for testing for t(9;22)(q34.1;q11.2) and BCR-ABL1; Recommendation for testing for KMT2A (MLL) translocations

· 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 includes, but is 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. Recommendation

· AML:

· For pediatric and adult patients with suspected or confirmed acute myeloid leukemia (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. Strong recommendation for testing for FLT3-ITD Recommendation for testing for other mutational analysis

· FISH:

· Unless the cytogenetic analysis is suboptimal because of poor chromosome morphology or insufficient cells for analysis or is completely unsuccessful because of no growth, FISH analysis may be an expensive, redundant technology, particularly in AML.

· In general, the utility of FISH should be considered in the context of each case

· For adult patients with confirmed core-binding factor (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. Strong recommendation for testing for KIT mutation in adult patients with CBF-AML Expert consensus opinion for testing for KIT mutation in pediatric patients with CBFAML

· For patients with suspected acute promyelocytic leukemia (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 (DIC). Strong recommendation

· The rapid diagnosis of APL is considered to be a medical emergency because of the risk of major hemorrhage, and MPO staining can be particularly helpful in cases of the microgranular variant of APL. It can be performed within 5 to 10 minutes and is available 24 hours a day, 7 days a week in many laboratories.

· The detection of MPO positivity in 3% or more of the blasts is a criteria for myeloid lineage delineation in AML.

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

· For patients with confirmed acute leukemia, no recommendation is made for or against the use of global/gene-specific methylation, microRNA (miRNA) expression, or gene expression analysis for diagnosis or prognosis. No recommendation

· For patients with confirmed mixed phenotype acute leukemia (MPAL), 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. Strong recommendation

· Laboratory Testing Site:

· All laboratory testing performed for the initial workup and diagnosis of a patient with acute leukemia must be performed in a laboratory that is in compliance with regulatory and/or accreditation requirements. Strong recommendation

· Reporting:

· 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. Strong recommendation

· 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. Strong recommendation Note.—This information should include the sample source, adequacy, and collection information, as applicable.

· Treating physicians and pathologists should use the current World Health Organization (WHO) terminology for the final diagnosis and classification of acute leukemia. Strong recommendation

References:

· CCO Dec. 2016 Consensus Pathology Recommendations for Complex Malignant Hematology

· Arber et al. Initial diagnostic workup of acute leukemia: guideline from the College of American Pathologists and the American Society of Hematology. Arch Pathol Lab Med 2017;141:1342-1393. (currently reading, at Statement 11)