Beyond the CBC Report: Extended Laboratory Testing in the Evaluation for Hematologic Neoplasia Disclosure

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1 Beyond the CBC Report: Extended Laboratory Testing in the Evaluation for Hematologic Neoplasia Disclosure I am receiving an honorarium from Sysmex for today s presentation. 1

2 Determining the Etiology for CBC Abnormalities In most circumstances, correlation with clinical parameters or results of other clinical tests will define the etiology for CBC abnormalities If hematopoietic neoplasia is considered as an etiology, extended laboratory testing requiring pathologist (MD) or PhD interpretation is required Examples of CBC Abnormalities Tied to Secondary Cause Abnormality Clinical Finding(s) Other Clinical Lab Test Results Diagnosis Microcytic Anemia Fatigue, pallor, shortness of breath Low serum iron and/or ferritin Iron deficiency anemia Macrocytic Anemia Pallor, shortness of breath Low vitamin B12 and/or folate levels Vitamin B12 or folate deficiency; pernicious anemia Erythrocytosis, leukocytosis Leukocytosis with neutrophilia Leukocytosis with lymphocytosis Chronic tobacco abuse Increased carboxyhemoglobin Secondary erythrocytosis related to smoking Cough, difficulty breathing, fever Sputum culture, chest X-ray Pneumonia Flu symptoms Influenza test Influenza Thrombocytosis Status post splenectomy Splenectomy related thrombocytosis Thrombocytopenia Easy bruising, bleeding Idiopathic thrombocytopenia purpura (ITP) 2

3 Examples of CBC Abnormalities Tied to Hematopoietic Neoplasia Abnormality Diagnosis Extended Testing Useful in Diagnosis Macrocytic anemia Myelodysplasia (MDS) Bone marrow evaluation, flow cytometry, cytogenetic karyotyping, SNP array Erythrocytosis Polycythemia vera (PV) Bone marrow evaluation, JAK2 mutation analysis Thrombocytosis Essential thrombocythemia (ET) Bone marrow evaluation, JAK2, CALR, and MPL mutation analyses Leukocytosis with leftshifted granulocytosis Myeloproliferative neoplasms (MPNs) such as chronic myelogenous leukemia (CML) Bone marrow evaluation, flow cytometry, FISH for BCR/ABL, cytogenetic karyotyping Circulating blasts Myelodysplasia or acute leukemia Bone marrow evaluation, flow cytometry, cytogenetic karyotyping molecular testing Lymphocytosis Lymphoid leukemia or peripheralized lymphoma Bone marrow evaluation, flow cytometry, cytogenetic karyotyping, FISH assays Hematopathology Pathology: the study of disease Hematopathology: subspecialty of pathology addressing diseases of blood and blood forming organs (blood, bone marrow, lymph nodes) Neoplastic hematopathology: study of neoplasias (cancers) that most often develop in blood, bone marrow, lymph nodes These diseases include leukemias, lymphomas, myelodysplasias, etc. 3

4 Hematopathologists Physicians with specialty training in pathology and subspecialty training in hematopathology In reference laboratories such as IO, hematopathologists are primarily concerned with neoplastic hematopathology We diagnose diseases (determine if disease is present or not and if present, we name it) For diseases that we diagnose, we are also involved in defining particular features relating to the prognosis of the disease Diagnosis: defines, classifies, or gives a name to the disease Prognosis: concerned with clinical behavior of the disease (benign, indolent, favorable prognosis, aggressive, poor prognosis) Techniques in Hematopathology Routine microscopy using histochemical and/or cytochemical stains Cytogenetic karyotyping (assesses for genetic abnormalities that are large enough to be visualized on a karyotypic spread) Immunohistochemistry (allows antigens expressed by cells to be visualized with a microscope) Flow cytometry: allows for efficient determination of antigenic profiles of cells in suspension; flow cytometer rather than the human eye detects the presence of antigens; analysis software then allows this to be evaluated by the human eye FISH (fluorescence in situ hybridization): allows detection of specific cytogenetic abnormalities, some of which can be seen in the karyotype and others that are too small to be seen in the karyotype Molecular assays (PCR, SNP array, NGS): sensitive assays that allow efficient detection of genetic abnormalities that FISH and cytogenetic karyotyping may fail to detect 4

5 Case Study 1: Leukocytosis with Lymphocytosis 82-year-old male with leukocytosis with lymphocytosis Oncologist requests evaluation for chronic lymphocytic leukemia (CLL) Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC H! K/uL NE% L % RBC L M/uL LY% H % HGB L g/dl MO% L % HCT L % EO% L % MCV fl BA% L % MCH pg NE# H K/uL MCHC L g/dl LY# H K/uL RDW H % MO# K/uL PLT K/uL EO# H k/ul MPV fl BA# K/uL 5

6 PBS Review PBS Review 6

7 Bone Marrow Evaluation Routine Stains Bone Marrow Evaluation Immunohistochemistry CD20 Cyclin D1 7

8 Flow Cytometry FISH Results 8

9 Final Diagnosis Leukemic mantle cell lymphoma Mantle cell lymphoma is an aggressive non- Hodgkin lymphoma; leukemic mantle cell lymphoma portends a worse prognosis than typical mantle cell lymphoma Typically involves lymph nodes and bone marrow Can present or progress to a leukemic form and this carries a particularly poor prognosis Mantle cell lymphoma must be distinguished from chronic lymphocytic leukemia Case Study 2: Leukopenia A 70-year-old female with leukopenia with neutropenia and mild normocytic anemia Negative clinical work-up 9

10 Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC L K/uL GRA% % RBC L M/uL LY% % HGB g/dl MID% % HCT % MCV H fl MCH H pg GRA# L K/uL MCHC g/dl LY# K/uL RDW % MID# K/uL PLT K/uL MPV fl Flow Cytometry Peripheral Blood 10

11 Flow QC Slide Review (Peripheral Blood) Bone Marrow Evaluation Routine Stains 11

12 Bone Marrow Evaluation Routine Stains Bone Marrow Evaluation Immunohistochemistry CD34 Hemoglobin A 12

13 Flow Cytometry Bone Marrow Cytogenetics Results Normal Karyotype 13

14 Molecular Testing Results NPM1 mutation assay: positive for mutation CEBPa mutation: negative FLT3 mutation: negative Summary of Risk Status in AML Based on Cytogenetics and Molecular Abnormalities Risk status Cytogenetics Molecular Better t(8;21), inv(16)/ t(16;16), t(15;17) Normal karyotype with NPM1 or CEBPA mutation and without FLT3 mutation Intermediate Normal, +8, t(9;11) t(8;21) or inv(16)/t(16;16) with c- KIT mutation Poor Complex, -5/5q, - 7/7q-, other 11q23, inv(3)/t(3;3), t(6;9), t(9;22) Normal karyotype with FLT3 mutation and without NPM1 mutation 14

15 Final Diagnosis AML with NPM1 mutation AML is an aggressive hematologic malignancy that progresses rapidly if left untreated The therapeutic approach and prognosis of AML is tied to genetic features and patient characteristics AML with NPM1 mutation, normal karyotype and absence of FLT3 mutation is a favorable prognostic form of AML Case Study 3: Anemia and Thrombocytopenia A 23-year-old male with pancytopenia (anemia, thrombocytopenia, and neutropenia) Atypical lymphocytes were noted on a peripheral blood smear 15

16 Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC K/uL NEU% L % RBC L M/uL LY% H % HGB L g/dl ESO% L 2.10 HCT L % BASO% % MCV fl MONO% % MCH pg NEU# L K/uL MCHC g/dl LY# K/uL RDW % ESO# K/uL PLT L K/uL BASO# K/uL MPV fl MONO# K/uL PBS Review 16

17 Bone Marrow Evaluation Routine Stains Bone Marrow Evaluation Immunohistochemistry CD10 TdT 17

18 Flow Cytometry Bone Marrow Cytogenetics Results Abnormal Karyotype Deletion of 9p21 with the loss of the CDKN2A gene, also known as p16 gene is observed in ~10% of childhood and ~30% of adult acute lymphoblastic leukemia (ALL) Deletion of the P16 gene is associated with a less favorable prognosis in pediatric B-lineage, but not in adult onset ALL or T-lineage ALL 18

19 FISH Results Deletion of CDKN2A gene, also known as p16 gene at 9p21, is observed in ~10% of childhood and ~30% of adult acute lymphoblastic leukemia (ALL) Deletion of the p16 gene is associated with a less favorable prognosis in pediatric B-lineage, but not in adult onset ALL or T-lineage ALL SNP Array Results The homozygous deletion of short arm 9 is specifically associated with T or B cell ALL with lymphomatous presentation and high white counts These cases have a higher propensity for mediastinal masses and T cell lineage Loss of the p16 tumor suppressor gene from this region is apparently involved in clonal selective advantage No consistent prognosis has been reported for ALL patients with p16 deletions 19

20 Final Diagnosis B-lymphoblastic leukemia/lymphoma (B-ALL) B-ALL must be distinguished from AML and T- ALL for therapeutic concerns Prognosis of B-ALL varies according to genetic alterations present Typical childhood B-ALL is highly curable with chemotherapy Subtypes with adverse genetic alterations are not as curable and may require bone marrow transplantation for cure Case Study 4: Pancytopenia A 53-year-old male with pancytopenia including significant neutropenia 20

21 Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC L K/uL NEU% L % RBC L M/uL LY% H % HGB L g/dl ESO% 0 2-5% HCT L % BASO% % MCV fl MONO% H % MCH H pg MCHC g/dl RDW % PLT L K/uL Bone Marrow Evaluation Routine Stains 21

22 Bone Marrow Evaluation Routine Stains Flow Cytometry Bone Marrow 22

23 Cytogenetics Results Abnormal Karyotype FISH Results Translocation (15;17) results in the fusion of the PML gene at 15q24 and the RARA gene at 17q21 and is characteristic of acute promyelocytic leukemia with t(15;17)(q24;q21); PML-RARA. 23

24 Final Diagnosis Acute promyelocytic leukemia (APL) APL is a subtype of acute myeloid leukemia (AML) with distinct morphologic, immunophenotypic, and genetic features Diagnosis confirmed by presence of PML/RARA genetic abnormality Diagnosis represents a medical emergency Typically responds to all trans-retinoic acid, a vitamin D derivative Case Study 5: Leukocytosis A 61-year-old female with leukocytosis and mild microcytic anemia Possible chronic myelogenous leukemia (CML) 24

25 Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC H K/uL GRAN% % RBC M/uL LY% % HGB L g/dl MID% % HCT L % GRAN# H MCV L fl LY# H MCH L pg MID# H MCHC g/dl RDW H % PLT K/uL Bone Marrow Evaluation Routine Stains 25

26 Bone Marrow Evaluation Routine Stains Bone Marrow Evaluation Immunohistochemistry CD61 CD34 26

27 Flow Cytometry Bone Marrow Cytogenetics Results 27

28 SNP Assay Results Test Performed Reveal (SM) SNP CMA - Oncology Specimen Type Bone Marrow Results # of Genotyping Targets: MICROARRAY DIAGNOSIS: 2.83 MB INTERSTITIAL DELETION OF 11Q12.3- >Q13.1; COPY-NEUTRAL LOSS OF HETEROZYGOSITY FOR CHROMOSOMES 3Q AND 22Q INTERPRETATION: CLONE DEMONSTRATING CLONAL EVOLUTION DETECTED Final Diagnosis Myelodysplastic/Myeloproliferative neoplasm: Atypical Chronic Myeloid Leukemia (acml) acml has both proliferative and dysplastic features and falls within the WHO category of MDS/MPN It must be distinguished from CML, which always can be demonstrated to have the Philadelphia chromosome and is effectively treated with tyrosine kinase inhibitors (typically imatinib) 28

29 Case Study 6: Pancytopenia A 65-year-old female with leukopenia, macrocytic anemia and thrombocytopenia Possible myelodysplastic syndrome Parameter Flag Result Reference Interval CBC Report Parameter Flag Result Reference Interval WBC L K/uL Neutrophil% % RBC L M/uL Lymphocyte% % HGB L g/dl Monocyte% H % HCT L % Eosinophil% % MCV H fl Basophil% % MCH H pg LUC% MCHC g/dl Neutrophil# L RDW H % Lymphocyte# PLT L K/uL Monocyte# Eosinophil#

30 PBS Bone Marrow Evaluation Routine Stains 30

31 Bone Marrow Evaluation Routine Stains Bone Marrow Evaluation Routine Stains 31

32 Flow Cytometry Bone Marrow Cytogenetics Results 32

33 SNP Assay Results Final Diagnosis Myelodysplastic Syndrome, Unclassifiable Diagnosis based on combination of morphologic, flow cytometric and SNP array findings As for classification, MDS, Unclassifiable and Refractory Cytopenia with Multilineage Dysplasia (RCMD) are possibilities, but MDS, Unclassifiable is the best fit given the subtle nature of the dyspoiesis 33

34 Summary The CBC report identifies blood abnormalities tied to neoplastic hematologic disorders that involve the blood These disorders are often first suspected based on CBC results in correlation with the clinical setting Diagnosis of these disorders requires involvement of pathologists Pathologists use several techniques to study blood and/or bone marrow (morphologic evaluation, immunohistochemistry, flow cytometry, cytogenetic karyotyping, FISH, molecular genetic tests) to define a diagnosis Thank You 34