Enhance Sensitivity of FISH Analysis with Highly Purified Multiple Myeloma Cells Using RoboSep, the Fully Automated Cell Separator

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1 Enhance Sensitivity of FISH Analysis with Highly Purified Multiple Myeloma Cells Using RoboSep, the Fully Automated Cell Separator Benoit Guilbault, PhD Field Applications Scientist t STEMCELL Technologies, Inc March 9th, 2012

2 Multiple Myeloma Background - Introduction Multiple Myeloma (MM) accounts for 10% of all hematological cancers Caused by the proliferation of a malignant plasma cell clone in the bone marrow Although can be treated using combinational therapy, there is no cure for this disease Photo Courtesy of Greg Ahmann, Mayo Clinic

3 Multiple Myeloma Background - Cytogenetic Analysis Oncogenic plasma cell transformation is caused by known genetic aberrations FISH remains the gold standard for the detection of these aberrations The nature of the genetic aberration can have a significant impact on patient prognosis Photo Courtesy of Ryan A. Knudson, Mayo Clinic

4 Multiple Myeloma Background Diagnostic Challenges Plasma cells can be extremely rare in bone marrow, making their detection difficult Plasma cells culture can also be a challenge due to a low proliferative index Genetic aberrations are therefore undetectable in a proportion of cases evaluated using standard techniques

5 Multiple Myeloma Background Diagnostic Challenges Whole Bone Marrow RoboSep -enriched enriched (CD138+) Photo Courtesy of Greg Ahmann, Mayo Clinic Performing cytogenetic analysis on a sample enriched for malignant plasma cells has been shown to increase assay sensitivity MM cells can be enriched by targeting the marker CD138 (Syndecan-1), which h is uniquely expressed on plasma cells

6 1. Add CD138 Selection Cocktail 2. Add magnetic nanoparticles Incubate 15 min. Incubate 10 min. 3. Pour out unwanted cells: CD138+ cells stay in the tube EasySep is a column-free immunomagnetic cell isolation system that is FISH and flow compatible

7 Benefits of using Tetrameric Antibody Complexes (TAC) Works at room temperature or 4 o C Stable several days on the bench EasySep Positively Selected Cells Long shelf-life (up to 2 years) Gentle on the cells Constant performance over time

8 EasySep is scalable Three magnet sizes available Sample size can range from <10 million to 4 billion start cells

9 Products for human cell separation T Cells (CD3 +, CD4 +, CD8 +, CD4 + CD25 + ) B Cells NK Cells Monocytes Granulocytes Plasma / Myeloma Cells (CD138) Mammary Cells (CD10 +, EpCAM +, MUC1 + ) Progenitor Cells

10 EasySep CD138 Positive Selection Kit Performance Bone marrow samples can be prepared either by Ficoll, or by washing and red cell lysis Sample size can range from 0.25mL Lto more than 10mL Typical EasySep result obtained from a PBMC sample spiked with a myeloma cell line

11 Case Study #1: EasySep -isolated CD138+ Cells Cytogenetics Laboratory The Ohio State University Medical Center Team: - Professor Nyla Heerema, PhD, ABMG,FACMG, Director of Cytogenetics - Carol Cole, BS, CG(ASCP), Lead Technologist - Jodi Hanna, BS, MSL(ASCP), Cytogenetic Technologist

12 Cell Isolation Protocol EasySep RBC Lysis Buffer EasySep CD138+ Selection Kit Average number of samples processed 21 samples per month Max number of samples processed 3 per day Time to validate ~1 month Number of samples to validate 15 samples in parallel Bone marrow (2mL) Plasma Cells Method of choice EasySep

13 Increased Detection of Genetic Aberrations in Separated vs. Unseparated Samples Unseparated Cells Separated Cells

14 Increased Detection of Genetic Aberrations in Separated vs. Unseparated Samples

15 The Frequency of Detected Genetic Aberrations per Sample is Increased in Separated vs. Unseparated Samples Unseparated Samples Separated Samples 6/15 abnormal 2%-33% abnormal cells 9/12 abnormal 7% -100% abnormal

16 Why Choose EasySep? 1. Ease of Use No need to babysit columns Just set the timer and keep working 2. Faster Protocol No more all day separations Start to finish in 1 hour 15 minutes 3. Accuracy of Results Greater comfort level Reliable results

17 EasySep can be fully automated with RoboSep

18 How Does RoboSep Work? Up to 4 samples at once The revolving carousel holds the g magnets, samples, reagents and empty tubes

19 How Does RoboSep Work? The cells are labeled and transferred to the magnet by the pipetting arm

20 How Does RoboSep Work? Filter tips are used One set of disposable tips per sample No cross-contaminationcontamination

21 How Does RoboSep Work? Select protocol Load samples, EasySep selection cocktail, magnetic particles, buffer and tips in carousel Press run RoboSep processes samples (approx min/run) Collect your separated cells

22 Benefits of using RoboSep to isolate cells for multiple myeloma research Saves technician time Processes 4 samples in about an hour with only 5 minutes of hands-on time Avoids sample contamination Samples handled independently using disposable tips to eliminate cross-contamination Stay safe All steps are fully automated to minimize sample handling True walk-away automation!

23 A NOVARTIS COMPANY Jelveh Lameh Ph.D. Dianne Keen-Kim, Ph.D. Amara Siva, Ph.D. Kristi Wolfe Farzad Nooraie, M.D Renee Mohrmann, M.D. Lee Kaplan, Ph.D.

24 Intelligent FISH for Myeloma (I-FISH) A NOVARTIS COMPANY Rationale: The pre-enrichment of plasma cells (CD138+ cells) prior to FISH is essential for samples with low-level plasma dyscrasias (<10% plasma cells) Goals of Study: To evaluate the clinical utility of enriching specimens for CD138+ plasma cells using immunomagnetic separation To report cytogenetic findings To compare these results to a matched sample of non-enriched specimens Source Clinical Data- Genoptix

25 Technical Requirements for Genoptix A NOVARTIS COMPANY High number of samples to process per day Requires automation Small volumes of bone marrow available Zero chance of cross contamination Must be able to process samples up to four days post-draw These samples often have decreased CD138 expression Source Clinical Data- Genoptix

26 Experimental Protocol A NOVARTIS COMPANY Remove 1 ml CD138 Positive Selection on RoboSep Assessment of Purity by FACS Whole Bone Marrow (3mL) FISH Analysis Source Clinical Data- Genoptix

27 Isolation of CD138 + cells with RoboSep A NOVARTIS COMPANY Unseparated Day 2 Day 3 Day 4 P2 = 13.4% plasma cells P2 = 92.3% P2 = 89.8% P2 = 75.9% CD38+/CD138+ CD38+/CD138+ CD38+/CD138+ cells cells cells Source Clinical Data- Genoptix

28 FISH images A NOVARTIS COMPANY FISH with IGH/FGFR: 80-90% trisomy FISH (cyto lab): 8.6% trisomy [26/300] Source Clinical Data- Genoptix

29 Genoptix Sample Processing A NOVARTIS COMPANY

30 A NOVARTIS COMPANY Distribution of Cytogenetic Aberrations Observed in Intelligent FISH for Myeloma Without Intelligent FISH With Intelligent FISH Source Clinical Data- Genoptix

31 Risk Stratification of Cases Assessed in Intelligent FISH for Myeloma A NOVARTIS COMPANY Assessment of cases before I-FISH Assessment of cases using I-FISH Source Clinical Data- Genoptix

32 Conclusions A NOVARTIS COMPANY RoboSep is capable of enriching plasma cells up to 4 days post-draw on as little as 1 ml bone marrow sample. The rate of cytogenetic abnormalities observed is dramatically higher in the enriched I-FISH cases (82%) than in the non-enriched specimens (28%). CD138-driven enrichment decreases the background of normal cells and increases the likelihood of observing a cytogenetic abnormality. Intelligent FISH can be used as an efficient and highly valuable technique for detection cytogenetic abnormalities in samples with lowlevel plasma cell levels Source Clinical Data- Genoptix

33 Discussion Section (Selected questions for full discussion, please view webinar). Q: Can the enriched CD138+ plasma cells be further cultured for chromosomal analysis? A: We don't have any information about being able to successfully culture BM cells post isolation. Keeping plasma cells in culture is very difficult to do. Dr. Heerema's group was unsuccessful in doing this. Q: Can you use EasySep and RoboSep on specimen types other than bone marrow? A. Yes, it works on any specimen where a single cell suspension can be achieved.

34 Discussion Section (Selected questions for full discussion, please view webinar). Q: Do you do any pretreatment t t on the cells before FISH? A: From Jodi (Dr. Heerema s lab manager): We treat the cells in a hyotonic solution for 10 minutes after separation, then prefix the cells before centrifugation and fixation.. Q: Do you separate all of the MM samples? Do you have a threshold? A. Yes, separate all samples. RoboSep can be used on samples with as little as 1% plasma cells (see Shetty et al. 2012: s and Leads