1. PLOIDY, AS DETECTED BY FLUORESCENCE IN SITU HYBRIDIZATION, DEFINES DIFFERENT SUBGROUPS IN MULTIPLE MYELOMA LEUKEMIA (2005) 19, 275–278. Keywords: myeloma, FISH, ploidy, DNA index, cytogenetic Dr Gihan Gawish 1 S Wuilleme 1, N Robillard 1, L Lodé 1, F Magrangeas 1, H Beris 2, J-L Harousseau 3, J Proffitt 2, S Minvielle 1 and H Avet-Loiseau 1 for the Intergroupe Francophone du Myélome 1 2 3 2 1 1 Laboratory of Hematology, University Hospital, Nantes, France 2 Vysis Inc., Downers Grove, IL, USA 3 Clinical Hematology Department, University Hospital, Nantes, France

2. Multiple Myeloma  Multiple myeloma MM, myeloma, plasma cell myeloma, or as Kahler's disease (after Otto Kahler) is a cancer of plasma cells.  plasma cells are a crucial part of the immune system responsible for the production of antibodies in humans and other vertebrates.  They are produced in the bone marrow and are transported through the lymphatic system. Due to the fundamental nature of the system affected, multiple myeloma manifests systemic symptoms that make it difficult to diagnose. Myeloma is generally thought to be incurable, but remissions may be induced with steroids, chemotherapy, thalidomide and stem cell transplants.  Myeloma is part of the broad group of diseases called hematological malignancies. Dr Gihan Gawish 2 Micrograph of a plasmacytoma, the histologic correlate of multiple myeloma

3. Multiple Myeloma Dr Gihan Gawish 3

4. Tow Cytogenetic Classification of Multiple Myeloma Dr Gihan Gawish 4 The two types of MM are approximately equally distributed

5. Dr Gihan Gawish 5  This cytogenetic classification is clinically valuable since patients with hyperdiploidy seem to present a better outcome than nonhyperdiploid patients.  However, this prognostic value has been demonstrated only in a few retrospective studies based on the analysis of karyotypes. Since cytogenetics is informative in only one-third of the patients, the prognostic significance of hyperdiploid vs nonhyperdiploid status in patients with uninformative karyotypes is not known.  To address this issue, other techniques have to be used that are not based on the requirement of clonal metaphases.  Flow cytometry has been proposed by several authors in order to evaluate the DNA index (DI). Nevertheless, no large study analyzing its prognostic value has been reported.  This cytogenetic classification is clinically valuable since patients with hyperdiploidy seem to present a better outcome than nonhyperdiploid patients.  However, this prognostic value has been demonstrated only in a few retrospective studies based on the analysis of karyotypes. Since cytogenetics is informative in only one-third of the patients, the prognostic significance of hyperdiploid vs nonhyperdiploid status in patients with uninformative karyotypes is not known.  To address this issue, other techniques have to be used that are not based on the requirement of clonal metaphases.  Flow cytometry has been proposed by several authors in order to evaluate the DNA index (DI). Nevertheless, no large study analyzing its prognostic value has been reported. Tow Cytogenetic Classification of Multiple Myeloma

6. Objective: Dr Gihan Gawish 6  Since bone marrow samples are often poor in MM and molecular cytogenetics is currently routinely performed at diagnosis, the ability of fluorescence in situ hybridization (FISH) to assess hyperdiploidy investigated.  In order to validate this approach, FISH and DI analyses in a series of 205 patients with MM was compared.

7. Patients, Materials and Methods: Dr Gihan Gawish 7 1- Patients and PC sorting  205 patients with MM were analyzed.  Patients with at least one million purified PC were included in this study.  There were 96 females and 109 males with a median age of 61 years (range=37–74).  A measure of 1–3 ml of heparinized bone marrow mononuclear cells were separated using Ficoll– Hypaque.  PC were then sorted using anti-CD138-coated magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Paris, France). PC purity was evaluated on Giemsa-stained cytospined cells.

8. Patients, Materials and Methods: Dr Gihan Gawish 8 2- Probe selection  In order to choose adequate probes for the assessment of hyperdiploidy, they started by systematically reviewing the cytogenetic literature focusing on the largest series.  In these reports, they specifically analyzed karyotypes with more than 48 chromosomes (with high hyberdiploidy) searching for the more frequently gained chromosomes.  Eight large published series have been reviewed hyperdiploid karyotypes.  They developed a specific probe cocktail for hyperdiploidy evaluation containing the LSI® D5S721/D5S23 labeled with SpectrumGreen TM, CEP®9 (alpha satellite) labeled with SpectrumAqua TM, and CEP®15 (alpha satellite, D15Z4) labeled with SpectrumOrang

9. Flowcytometry and FISH: Dr Gihan Gawish 9 Probes specific for t(4;14)(p13;q32), t(11;14)(q13;q32), and del(13) (LSI D13S319) were furnished by Abbott/Vysis and used according to standard recommendations. Flow cytometry analysis was performed on a FACSCalibur using CELLQuest Pro software (BD- Bioscience) using propidium iodide

10. DI = (ratio of the mean or peak of sample G0/G1 population divided by the mean or peak of diploid reference cells). DNA diploid PC were defined as containing a single G0/G1 population presenting a DI=1.0 DNA Index calculations Dr Gihan Gawish 10

11. Results: Dr Gihan Gawish 11 (a) A typical profile corresponding to a patient with a diploid clone. Only one peak is observed. (b) The same patient hybridized with the ploidy probe. Two green, two red, and two white signals corresponding to chromosomes 5, 9, and 15, respectively, are observed. (c) A typical profile obtained in a hyperdiploid patient. The first peak corresponds to the normal cells, whereas the second peak corresponds to the myeloma cells. The DI was calculated to 1.32. (d) The same patient hybridized with the ploidy probe, showing three signals with each probe, and thus confirming hyperdiploidy.

12. Correlations between flow cytometry and FISH experiments Dr Gihan Gawish 12 Diploid by FISH Hyperdiploi d by FISH Tetraploid by FISH t(11;14)t(4;14)del(13q14) DI<17 323 DI=1 − 1.04 91 421761 DI=1.05 − 1.5 698 7750 DI>1.6 31 2 DI=DNA index, measured by flow cytometry; FISH=fluorescence in situ hybridization.

13. Conclusion Dr Gihan Gawish 13  In this study utilizes a novel FISH test to separate myeloma into hyperdiploid and nonhyperdiploid groups.  The nonhyperdiploid group is tightly associated with recurrent 14q32 translocations and to a lesser degree with del(13).  As this test is performed in interphase, it is independent of the proliferation rate.  This test will permit the evaluation of the prognostic value of ploidy in myleoma without the need for karyotyping and is currently being used for the patients enrolled in the IFM 99 therapeutic trials.  These studies should enable to address the issue of the true prognostic value of ploidy per se.  Actually, the reports addressing these questions have been performed by conventional cytogenetics, thus in patients with a high proliferation. Whether the prognostic value is associated mostly to ploidy or mostly to proliferation is an interesting question.

14. Summary Dr Gihan Gawish 14  Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma.  However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma).  A novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients was validated.  The method was very specific and sensitive for the detection of hyperdiploidy.  Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%).  Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.  Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma.  However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma).  A novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients was validated.  The method was very specific and sensitive for the detection of hyperdiploidy.  Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%).  Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.