Cost-Effective Strategies in the Workup of Hematologic Neoplasm. Karl S. Theil, Claudiu V. Cotta Cleveland Clinic
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1 Cost-Effective Strategies in the Workup of Hematologic Neoplasm Karl S. Theil, Claudiu V. Cotta Cleveland Clinic
2 In the past 12 months, we have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in the presentation.
3 Goals Review opportunities for appropriate laboratory testing in MPN, MDS, AML and NHL. Identify redundancies and inefficiencies in utilization of laboratory tests. Advise clinicians on the rational utilization of laboratory tests.
4 Choosing Wisely Help physicians and patients engage in conversation to reduce overuse of tests and procedures, and support physician efforts to help patients make smart and effective care choices.
5 PubMed Search on Cost Effectiveness Number of Articles Publication Year
6 PubMed Search on Cost Effectiveness
7 Google Search on Cost Effective
8 Brain-to-Brain Turnaround Time Loop Lundberg GD: Acting on significant laboratory results. JAMA 1981; 245 (17):
9 Brain-to-Brain Turnaround Time Loop Am J Clin Pathol 2011; 136(6):
10 Test Requisition
11 Methods and Assumptions Assumptions: The location of the test (in-situ vs. reference lab) is not a significant criterion in the selection of tests The prices reflect the costs All the tests are reimbursed at the same rate Methods: Prices from several major reference labs were compared The quantitative considerations were made starting from the average price
12 Caveats Our focus is on the role of the pathologist in test triage. Our practice model may not be generalizable. Evaluating clinical effectiveness requires a partnership with clinicians.
13 Myeloproliferative Neoplasms Specimens to work with: Peripheral blood Bone marrow aspirate and biopsy Tissue Information to be obtained: Diagnosis Prognosis
14 MPN- Tests to Be Performed CBC with differential Bone Marrow Evaluation with reticulin (0.5 $) Fluorescence in situ hybridization (5 $) Molecular studies: BCR-ABL, JAK2 (5 $) Cytogenetics (5 $)
15 Common Scenarios Flow cytometry requested on new diagnosis of chronic myelogenous leukemia JAK2 and BCR-ABL ordered simultaneously or not at all BCR-ABL FISH and RT-PCR requested together or not at all
16 Algorithmic Approach BCR-ABL rearrangement is disease-defining for CML and it must be excluded in order to consider diagnosis of other MPN. JAK2 mutation is frequent (>95%) in PV and common in ET and PMF (~50%). Molecular analysis for BCR-ABL and JAK2 have a central role in the initial diagnosis of MPN.
17 JAK2 Testing JAK2 V617F mutation or exon 12 mutations are present in virtually all cases of PV and are mutually exclusive. Indications include unexplained polycythemia, neutrophilia or thrombocytosis, splanchnic vein thrombosis or RARS-T. Test for JAK2 V617F first; if negative, test for exon 12 mutation. J Mol Diagn 2013; 15(6): 1-12 [Epub ahead of print].
18 JAK2 Testing Peripheral blood and bone marrow are equivalent samples. Methods include allele-specific qpcr ( 1% mutant alleles) and melt curve analysis (5-10% mutant alleles). Screen first with V617F mutation and restrict exon 12 mutation testing to a reflex test. J Mol Diagn 2013; 15(6): 1-12 [Epub ahead of print].
19 MPN- Resource-Saving Strategies Time (and $$) saving tools: Triage requests after review of blood smear (and bone marrow) Flow cytometry not helpful in chronic phase CML Obtain specimens for cytogenetics (blood vs. marrow) and can use remainder of cytogenetics pellet for FISH Obtain specimens for DNA/RNA extraction and hold for future testing
20 Leukemias Specimens to work with: Peripheral blood Bone marrow aspirate Bone marrow aspirate smears (or touch imprints) Aspirate clots Bone marrow cores Tissue (in cases of myeloid sarcomas/lymphoblastic processes)
21 Leukemias Information to be obtained: Diagnosis Lineage Phenotype Diagnostic cytogenetic abnormalities Prognosis Cytogenetics Molecular
22 Leukemias-Tests to Be Performed Morphologic examination-wg, HE (1 $) Cytochemical stains (0.5 $) Flow cytometry (15 $) Immunohistochemistry (1 $) Fluorescence in-situ hybridization (4 $) Conventional cytogenetics (5 $) PCR (including limited sequencing) (3 $) Chromosomal/SNP microarrays (8 $)
23 Leukemias The workup is almost always divided in two stages: initial and final. The initial workup should focus on establishing the diagnosis of leukemia, clarifying the lineage, the presence of PML/RARA. The final workup should result in a diagnosis according to the WHO 2008 classification, including cytogenetic and molecular information.
24 Leukemias-Initial Workup Specimens necessary: Peripheral blood Bone marrow. In many cases clinicians prefer to perform a bone marrow biopsy even if there are sufficient blasts in the peripheral blood. Tests to be performed: Morphologic examination, including, when necessary cytochemical stains (2 $) Flow cytometry (15 $) FISH/PCR for PML/RARA and BCR/ABL (4 $ each) Cytogenetics (5 $) Nucleic acid extraction (0.5 $)
25 Identification of t(15;17)/pml-rara Methods available: Cytogenetics, FISH, RT-PCR FISH: break-apart probes for RARA and fusion probes for PML-RARA quick, 5 hour incubation protocols exist If a choice is necessary, the break-apart probes are recommended RT-PCR: may be less effective and it may take too long for the initial diagnosis, but it is optimal for the detection of minimal residual disease Given the clinical consequences, probably the deciding factors in test selection are time and sensitivity Burnett AK, et al. Blood 1999;93: Mancini M, et al. Br J Haematol 1995;91: Fukutani H, et al. Leukemia 1995;9:
26 Identification of PML-RARA RT-PCR
27 t(15;17) by Conventional Cytogenetics
28 Myeloid Leukemias-Initial Workup Morphology Flow Cytometry Initial Workup Conventional Cytogenetics FISH or RT-PCR for RARA (optional) Nucleic Acid Extraction
29 Myeloid Leukemias-Final Work-up Goal: diagnose the neoplasm according to the WHO 2008 classification: Acute myeloid leukemia with recurrent genetic abnormalities Acute myeloid leukemia with gene mutations- NPM1 and CEBPA
30 Pie chart based on 246 patients with complete data sets indicating the mutation status coexisting in individual patients for the NPM1, FLT3 (ITD and D835), CEBPA, and MLL (PTD) genes. Döhner K et al. Blood 2005;106: by American Society of Hematology
31 Myeloid Leukemias-Final Workup Specimens necessary Nucleic acids Cytogenetic pellets Aspirates/Peripheral Blood smears Tests to be performed-mainly identification of mutations with probable prognostic significance Sequencing FISH Chromosomal/SNP microarrays
32 Myeloid Leukemias-Final Workup Nucleic acid extraction at the time of the initial workup and the results of conventional cytogenetic analysis allow for a rational utilization of resources. Tests for NPM1, FLT3, CEBPA mutations are not necessary in cases with cytogenetic abnormalities. Additional tests for IDH1, IDH2, TET2, ASXL1, DNMT3a, MLL-PTD can be ordered later, if the clinicians request them. Tests for c-kit mutations should only be ordered in cases with t(8;21) or inv(16).
33 Myeloid Leukemias-Final Workup Inv(16) or t(8;21) Extracted Nucleic Acids Conventional Cytogenetics Abnormal c-kit mutations (3$) Diploid Extracted Nucleic Acids Chromosomal/SNP microarrays (8$) NPM1, FLT3, CEBPA mutations (7$)
34 Detections of NPM1 Mutations
35 Lymphoblastic Leukemia-Final Workup Cytogenetics FISH: Chromosomes 4, 10, 17 (COG panel), mainly for pediatric cases. BCR/ABL
36 Leukemias: Resource-Saving Strategies Use nucleic acid extraction on all de-novo specimens Final work-up should be performed only after the cytogenetic data is available and the additional tests should be ordered on the extracted nucleic acids The utility of microarrays is still debatable and their performance compared to that of conventional cytogenetics or molecular techniques has not been widely investigated
37 Myelodysplastic Syndromes Specimens to work with: Peripheral blood Bone marrow aspirate/smears Aspirate clot section Bone marrow biopsy/touch imprint
38 Myelodysplastic Syndromes Information to be obtained: Establish Diagnosis Is the diagnosis really MDS or something else? Establish WHO Classification Cytopenia(s), dysplasia, % blasts, cytogenetics Determine Prognosis IPSS-R Score depends on % blasts and cytogenetics
39 MDS- Tests to Be Performed CBC with differential and smear review (0.5 $) Bone marrow aspirate and biopsy (5 $) Chemistry tests (ferritin, B12, folate, copper) (2 $) Flow cytometry (15 $) Immunohistochemistry (1 $) Fluorescence in situ hybridization (4 $) Cytogenetics (5 $) Chromosomal/SNP microarrays (8 $)
40 Common Scenarios Level of clinical suspicion varies and influences specimens obtained/sent for testing Incomplete clinical information available to pathologist FISH for MDS and cytogenetics requested simultaneously No sample obtained for molecular studies
41 MDS- Flow cytometry Guidelines proposed by European LeukemiaNet Working Group to meet minimal diagnostic criteria for MDS Technical and standardization issues and establishment of normal variation Antigen aberrancy may precede morphologic dysplasia. Leukemia 2012; 26(7): Leukemia & Lymphoma 2013; Early Online 1-12.
42 MDS- Immunohistochemistry May be helpful in cases suspected or provisional for MDS. Recommended markers: CD34, CD117, tryptase, CD61 or CD42b, CD20, CD3, glycophorin A or C. Additional markers as needed if MDS diagnosis is in question or other neoplasm is suspected. Oncotarget 2010; 1:
43 MDS- Cytogenetics vs. FISH Identification of clonal chromosome abnormalities facilitates diagnosis and classification Clonal changes are not pathognomonic without evidence of dysplasia: trisomy 8, del(20q) Clonal changes may be required for diagnosis and classification: 5q- syndrome (sole abnormality) FISH complements cytogenetic analysis
44 Am J Clin Pathol 2011; 135(6):
45 46,XY,-7,del(9)(q22q34),del(20)(q11.2),+mar
46 46,XX,+1,der(1;7)(q10;p10)
47 MDS- Cytogenetics vs. FISH FISH studies added little to the diagnostic yield of adequate metaphase cytogenetics alone. MDS FISH panel testing should be reserved for cases with suboptimal growth or when clarification of an abnormal karyotype is indicated. Low-level clones detected by both methods. Am J Clin Pathol 2011; 135(6):
48 MDS- Resource-Saving Strategies Triage requests after review of peripheral blood smear and bone marrow aspirate Use electronic medical record to obtain missing/incomplete data Use FISH as an adjunct to metaphase cytogenetics Share samples between laboratories to obtain specimens for DNA/RNA extraction and hold
49 Lymphomas Specimens to work with: Tissue (including Bone Marrow biopsy core) Cell suspension (including aspirate) Slides with smears Information to be obtained: Diagnosis Prognosis
50 Lymphomas-Tests to Be Performed Cytologic examination: H&E, Wright-Giemsa, DiffQuik (1 $) Histologic sections: H&E (0.5 $) Flow cytometry (10-15 $) Immunohistochemistry (1 $) Fluorescence in-situ hybridization (FISH) (4 $) Polymerase chain reaction: BIOMED-2 (4 $) Southern blot (3 $) Conventional Cytogenetics (5 $)
51 Lymphomas-Flow Cytometry Specimens should be collected in every case when lymphoma is suspected. In many cases, the flow cytometry specimen can be put on hold, until the histologic sections are examined (next day). Given the equation 1 lymphoma flow pannel = stains, in cases with straightforward features flow cytometry may not be necessary.
52 Examples When Flow Cytometry May Not Be Necessary Chronic lymphocytic leukemia/small lymphocytic lymphoma Follicular lymphoma Diffuse large B-cell lymphoma Classical Hodgkin lymphoma Nodular lymphocyte predominant Hodgkin lymphoma Anaplastic large cell lymphoma, ALK-positive Etc.
53 Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma HE CD20 CD5 Lef1 Details on LEF1 in Tandon B., et al. Mod. Pathol. 2011, Nov;24(11):
54 Lymphomas-Immunohistochemistry Decisions to be made: Individual stains vs. Panels (time vs. money) Flow cytometry vs. Immunohistochemistry Prognostic/Theragnostic markers (CD30, c-myc, ABC vs GC, Ki-67, etc.) IHC screening before ordering FISH (IGH/BCL2, Cyclin D1, MYC, ALK, etc.) Unstained slides to be used later, if necessary, to save tissue, time
55 Lymphomas-Immunohistochemistry Short panels for lymphomas, when flow not available (or not deemed necessary): Chronic lymphocytic leukemia/small lymphocytic lymphoma: CD3, CD20, LEF1 Follicular lymphoma: CD10, CD20, CD21, BCL-2, BCL-6 Mantle cell lymphoma: CD3, CD20, Cyclin D1
56 Lymphomas-Immunohistochemistry Prognostic markers: know what the clinicians want. In mantle cell lymphoma: Ki-67, Sox11. FISH for CLL/SLL In diffuse large B-cell lymphoma: CD10, CD30, BCL-2, BCL-6, Ki-67, MUM1, c-myc. IHC for BCL-2 and c-myc is a great tool in screening for translocations involving BCL-2 and MYC, (1$ vs 4$).
57 Screening for cases positive for translocations involving MYC Details regarding correlations between IHC stains for MYC and translocations involving MYC in Ruzinova MB, et al. in Am. J. Surg. Pathol. 2010, Jun;34(6):
58 Lymphomas-Molecular tests Often not necessary when a clone or an abnormal immunophenotype can be demonstrated by morphology/ihc/flow cytometry. In terms of cost, 1 flow cytometry panel = 5 x IHC + 1 PCR Flow cytometry and PCR-based tests are relatively equally sensitive in the detection of clonal B or T-cell populations. Dictor M. et al. In Clin Chem Lab Med 2007;45(3): Morice W.G. et al. In Am J Clin Pathol 2004;121(3):
59 Lymphomas-Flow Cytometry vs. Molecular tests
60 Lymphoma-Conclusions Time (and $$) saving tools: Flow cytometry to be performed after morphologic examination IHC for BCL2 and c-myc Unstained slides Minimize overlap between flow cytometry and IHC
61 Conclusions The pathologist can play a critical role in guiding laboratory test utilization to optimize cost effectiveness and clinical efficiency. This requires close communication and cooperation with clinicians. Effective use of electronic medical record can facilitate test ordering and reporting.
62 Conclusions Developing consistent testing strategies helps ensure that the right test is performed on the right patient at the right time. Avoids over-testing and under-testing
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