6/20/2014. PART I: Plasma Cell Myeloma. Plasma Cells

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1 MULTIPLE MYELOMA: THE TESTING, VALIDATION AND IMPLEMENTATION OF CELL SEPARATION TECHNOLOGY FOR IMPROVED PATIENT CARE Elizabeth Harper CG(ASCP), Binh Vo CG(ASCP), Joey Pena CG(ASCP), Denise Lovshe CG(ASCP), Dr. Gary Lu, Dr. Xinyan Lu PART I: Plasma Cell Myeloma The major disease of plasma cell neoplasms Results from the expansion of a clone of immunoglobulin (Ig)-secreting, heavy-chain classswitched, terminally differentiated B cell (plasma cell) Typically secrets a monoclonal immunoglobulin called paraprotein or M-protein The 2 nd most prevalent hematologic neoplasm worldwide 4.3/100,000; 15,000 cases/year in USA; twice as common in blacks Clonal plasma cell expansion Plasma Cells Abundant blue cytoplasm Pale perinuclear zone Round eccentric nucleus, spoke wheel or clock face chromatin without nucleoli May present with multiple nuclei 1

Xinyan Lu PART I: Plasma Cell Myeloma The major disease of plasma cell neoplasms Results from the expansion of a clone of immunoglobulin (Ig)-secreting, heavy-chain classswitched, terminally

2 Bone Marrow Evaluation Plasma cell count: >30% in BM (if <30%, tumoral mass of plasma cells displacing normal BM elements) Usually occur in interstitial clusters, focal nodules or diffuse sheets Early stage: interstitial and focal Advanced stages: diffuse and or extra-medullary involvement Plasmablastic morphology as a prognostic factor: Immaturity and pleomorphism are reliable indicators of malignant plasma cells A higher percentage of BM plasma cells and higher plasma cell labeling index higher proliferation rate Significant lower response rate Other Markers Elevated β-microglobulin Increased serum calcium Urine protein Bone lesion Immunohistochemistry (IHC) Staining Useful in quantifying plasma cells on biopsy Useful in confirming a monoclonal plasma cell proliferation CD138 stain: useful for quantifying plasma cells, and clonality can usually be established with stains for Kappa and Lambda light chains Useful in distinguishing myeloma from other neoplasms 2

malignant plasma cells A higher percentage of BM plasma cells and higher plasma cell labeling index higher proliferation rate Significant lower response rate Other Markers Elevated β-microglobulin

3 Flow Cytometry Analysis Diagnostic markers CD138 expression, CD38 strong expression, and absence to dim CD45 Prognostic markers CD56 higher incidence of extramedullary disease Higher β2-microglobulin Plasmablastic morphology CD44v9: poor prognosis CD20: more aggressive MM subtype, shorter survival CD27: significantly higher in MM remission The Common 4 Cytogenomic Pathways For Myeloma GERMINAL CENTER B-CELL MGUS Aneuploidy (Chromosome gains) IGH/CCND1 XT TP53 hypodiploid, RAS, p18ink4c, others Smoldering Myeloma IGH rearrangements Medullar Myeloma -13/del(13q) Extramedullar Myeloma IGH/FGFR3 Chromosome 1 rearrangements c-myc PCL Aneuploidy Pathway Almost 100% by DNA content analysis by flow cytometry Most are primary Hyperdiploidy in most cases Non-hyperdiploidy 3

For Myeloma GERMINAL CENTER B-CELL MGUS Aneuploidy (Chromosome gains) IGH/CCND1 XT TP53 hypodiploid, RAS, p18ink4c, others Smoldering Myeloma IGH rearrangements Medullar Myeloma -13/del(13q)

4 Plasma cell myeloma (PCM) Monoclonal gammopathy of Undetermined significance (MGUS) 1% IGH Pathway In about 45% MM Involves at least 27 different chromosome partners The most common partners (> 2%): 4, 6, 11, 16, and 20 Diverse prognosis depending on partner involved IGH Rearrangement (1) t(11;14)(q13;q32) 15-20%/FISH in MM; 15-30% in MGUS Involving CCND1 Favorable prognosis if without risk markers 4

4, 6, 11, 16, and 20 Diverse prognosis depending on partner involved IGH Rearrangement (1)

5 IGH Rearrangement (2) t(4;14)(p16;q32) 15-20% by FISH Involving FGFR3 The most Unfavorable prognosis IGH Rearrangement (3) t(14;16)(q32;q23) 2-10% by FISH Involving MAF Unfavorable prognosis 5

6 IGH Rearrangement (4) t(6;14)(p21;q32) 3-5% by FISH Involving CCND3 Unfavorable prognosis del(13q)/-13 Pathway 40%-50% by FISH Frequently present w/ t(4;14), t(14;16) and +1q scattered along the long arm by acgh Increased frequency with increased plasma cell bone marrow load Unfavorable prognosis 6

present w/ t(4;14), t(14;16) and +1q scattered along the long arm by acgh

7 Plasma Cell Enrichment Use of CD138 Magnetic MicroBeads: Targets CD138 antigen, a transmembrane heparin sulfate proteoglycan macromolecule CD138+ cells include normal and malignant human plasma cells Not expressed on virgin/naïve B cells, memory B cells, T cells, or monocytes Plasma Cell Enrichment Incubation with CD138 Magnetic MicroBeads to target plasma cells After incubation cells suspension is passed through a Column located in the Magnet Separator The Column is removed from Separator and Elution Buffer is used to flush out magnetically labeled (CD 138+) plasma cells CD38 Immunofluorescent Staining CD38 antigen is a transmembrane glycoprotein with enzymatic activity, high expression on plasma cells Incubate cells with CD38-Biotin antibody, followed by a wash and incubation with anti-biotin-fitc antibody 200 cells scored pre- and post- plasma cell enrichment 7

is passed through a Column located in the Magnet Separator The Column is removed from Separator and Elution Buffer is used to flush out magnetically labeled (CD 138+) plasma cells CD38

8 CD38 Immunofluorescent Staining Pre-PCE Case 1 Case 2 7.5% 3.2% Post-PCE 42% 89% PC Enrichment Factor PC Enrichment Factor PC count from Aspirate Smear by Morphology assessment Range: 0-63% Median: 2.6% PC % Pre-PCE: 1-18% Post-PCE: 31-93% Enrichment Factor Range: 4-52 Median:

from Aspirate Smear by Morphology assessment Range: 0-63% Median: 2.

9 PART II: CURRENT PROCESSING ALGORITHMS Bone marrow sample arrives to lab. Separate all charts with Myeloma diagnosis and with FISH order * Take 2 blue falcon tubes and label them with Patient name and MR# * Invert the syringe to mix, then transfer 1ml of bone marrow into one labeled (Patient Name; MR#) blue tube and the remaining bone marrow into a second labeled (Patient Name; MR#)blue tube. BM FISH CULTURE PROCESSING * Add media, order the BM24-X label and set up the routine specimen with the bone marrow in the blue falcon tube. * If there is extra bone marrow after setting up, record as + sign in volume column of the RoboSep log, next to 1ml. ROBOSEP SAMPLE PROCESSING * Add 2 ml s of media to the 1ml of bone marrow and place in Myeloma Specimens canister in tc room fridge. * Record specimen information on log; next day determine the plasma cell count in Clinic Station; located in Pathology folder. 9

one labeled (Patient Name; MR#) blue tube and the remaining bone marrow into a second labeled (Patient Name; MR#)blue tube.

10 ROBOSEP SAMPLE PROCESSING * If the plasma cell concentration is between 3-15%, process specimen using RoboSep for plasma cell separation. * Obtain the enriched plasma cells after the RoboSep process. * Remove RoboSep tubes; shake gently to dislodge PC s from sides of tube and refrigerate ROBOSEP SAMPLE PROCESSIING Perform RoboSep Shutdown Procedure Centrifuge plasma cells and harvest according to procedure. (may be performed immediately or up to 1 day post-processing). RoboSep harvester responsible for notifying routine BM harvesters that Biogenix slides are available. MYELOMA FISH ALGORITHM 10

* Remove RoboSep tubes; shake gently to dislodge PC s from sides of tube and refrigerate ROBOSEP SAMPLE PROCESSIING Perform RoboSep Shutdown Procedure

11 PART III: ROBOSEP PROCESSING, SUPPLIES AND QC Isolation of specimens to be RoboSeped (parameters, location of storage) Preparing samples for RoboSep (buffer wash; lysis buffer) Preparing supplies; readying machine Loading the RoboSep and selecting protocols PART III: PROCESSING THE SAMPLE AND LOADING THE ROBOSEP May use any number of stations up to 4 Machine gives quantity of supplies needed as the selection process for each sample continues Machine runs for 53-1:10 minutes depending upon quantity of samples loaded PART III: LOADING THE ROBOSEP 11

PROCESSING THE SAMPLE AND LOADING THE ROBOSEP May use any number of stations up to 4 Machine gives quantity of supplies needed as the

12 PART III: LOADING THE ROBOSEP PART III: ROBOSEP SUPPLIES PART III: ROBOSEP QC 12

13 PART IV: ROBOSEP HARVESTING VALIDATION Over 175 Myeloma patient specimens processed Initial processing performed on plasma cell counts ranging from 0-15% Early attempts to achieve optimal cell count has led to much refinement of what to process and the harvesting procedure. PART IV: ROBOSEP HARVEST VALIDATION Harvester drops suspension on BioGenex slides using micropipettor The harvester checks the quality of the slides and determines which patient slides are acceptable The patient with acceptable slides has their slide condition changed from reserve to epc PART V: COMPARISON AND VALIDATION OF FISH PROBES Myeloma Panel CCND1/IGH-XT t(11;14) RB1 (13q14) P53 (17p13.1) CKS1B (1p32.3 & 1q21) 10 samples for each probe 13

PART IV: ROBOSEP HARVEST VALIDATION Harvester drops suspension on BioGenex slides using micropipettor The harvester checks the quality of the slides and determines which patient

14 CCND1/IGH-XT PROBE ROBOSEP % POSITIVITY BM FISH % POSITIVITY RB-1 PROBE ROBOSEP % POSITIVITY BM FISH % POSITIVITY TP53 PROBE ROBOSEP % POSITIVITY BM FISH % POSITIVITY 14

15 CKS1B PROBE ROBOSEP % POSITIVITY BM FISH % POSITIVITY PART VI: REPORTING ROBOSEP SAMPLE RESULTS New phrase codes: EPC ENRICHED PLASMA CELLS FROM BONE MARROW 27173: The enriched plasma cells used in this study are derived from bone marrow specimen using immunomagnetic cell separation technology, which increases FISH sensitivity for detecting cytogenetic abnormalities associated with myeloma. PART VII: VALIDATION STUDIES New procedure requires new analytic validation of all the myeloma panel probes using 20 negative control samples to establish a normal reference range or cutoff. Currently working on getting the data and eventually performing the statistical analysis to derive the cutoff value for each of the 4 probes currently in the MM panel. 15

16 ACKNOWLEDGEMENTS Thanks to Dr. Lu for bringing this technology to the lab Thanks to Denise for securing the RoboSep lease Thanks to Stem Cell for training and continued support ROMEO AND JULIET Thanks to the lab for adapting a new technology Thanks to the Technicians who set aside 175+ and counting samples for RoboSep processing Thanks to the XMatrx Team for their hard work towards integration Thanks to all RoboSep Team members: past (Aruna, Jun), present, and future! 16

training and continued support ROMEO AND JULIET Thanks to the lab for adapting a new technology Thanks to the

FastTest. You ve read the book... ... now test yourself

FastTest. You ve read the book... ... now test yourself FastTest You ve read the book...... now test yourself To ensure you have learned the key points that will improve your patient care, read the authors questions below. Please refer back to relevant sections