Presentation on theme: "CYTOGENERIC ASSESSMENT IN MDS"— Presentation transcript:
1 CYTOGENERIC ASSESSMENT IN MDS NOTE: These slides are for use in educational oral presentations only. If any published figures/tables from these slides areto be used for another purpose (e.g. in printed materials), it is the individual’s responsibility to apply for the relevant permission. Specific local use requires local approval.
2 Outline MDS Classification and prognosis scoring Practical guide to bone marrow aspirate analysis in MDSCytogenetics of MDS in bone marrow aspiratesSummaryLIC = liver iron concentration; MRI = magnetic resonance imaging;SF = serum ferritin; SIR = signal intensity ratio;SQUID = superconducting quantum interface device.
3 What are the myelodysplastic syndromes (MDS)? MDS are a spectrum of heterogeneous myeloid clonal disordersOccurrence:De novo (primary MDS)Secondary or treatment-related MDSMDS are associated with significant morbidity and mortality due to:Risk of transformation to acute myeloid leukemia (AML)CytopeniasImpaired quality of life (frequent transfusions, iron overload, etc)3
4 Common features of MDSMDS are marked by ineffective haematopoiesis and defective development of blood cells, e.g.:dyserythropoiesis (affects red blood cells production)dysgranulopoiesis (affects granulocytes production)dysmegakaryopoiesis (affects platelet production)Ineffective haematopoiesis and maturation of the blood cells results in one or more cytopenias, e.g.:anaemia (reduced red blood cell count)neutropenia (reduced absolute neutrophil count)thrombocytopenia (reduced number of platelets)
5 Minimal diagnostic criteria in MDS consensus, Vienna 2006 Prerequisite criteriaconstant cytopenia in one or more cell lineagescomplete blood countexclusion of all other causes of cytopenia / dysplasiaMDS-related (decisive) criteriadysplasia in > 10% of all bone marrow cells in one or more of the lineages, or > 15% ringed sideroblastsiron staining of bone marrow smears5–19% blast cellsbone marrow smearstypical chromosomal abnormalitykaryotyping or FISHFISH = fluorescence in situ hybridization.Loken MR, et al. Leuk Res. 2008;32:5-17. Nimer SD. Blood. 2008;111: Valent P, et al. Leuk Res. 2007;31:
6 Minimal diagnostic criteria in MDS consensus, Vienna 2006 (cont.) Additional criteria (for patients not fulfilling the decisive MDS criteria)abnormal phenotype of bone marrow cellsflow cytometrymolecular signs of a monoclonal cell populationHUMARA assay, gene chip profiling, point mutation or SNP analysismarkedly and persistently reduced colony formationCFU assayCFU = colony-forming unit; SNP-a = single-nucleotide polymorphism.Loken MR, et al. Leuk Res. 2008;32:5-17. Van de Loosdrecht AA. Leuk Res. 2008;32: Van de Loosdrecht AA, et al. Blood. 2008;111: Van de Loosdrecht AA, et al. Haematologica. 2009;94: Nimer SD. Blood. 2008;111: Valent P, et al. Leuk Res. 2007;31:
8 Diagnostic and prognostic of MDS Classification systemBasis of evaluationDerived prognostic score systemFAB (1982)Cellular morphologyIPSS (1997)WHO (2002)Cellular morphology, cytogeneticsWPSS (2007)Risk-stratification is necessary for clinical decision-making:predicts treatment outcomespredicts survivalpredicts risk of progression to AMLFAB, French-American-British; IPSS, International Prognostic Scoring System; WPSS, WHO-based Prognostic Scoring System.Bennett JM, et al. Br J Haematol 1982;51:189–199; Jaffe, et al, eds. Lyon: IARC Press; 2001; Greenberg P, et al. Blood 1997;89:2079–2088; Malcovati L, et al. J Clin Oncol 2007;23:3503–3510.8
9 French-American-British (FAB) classification Blast percentageMDS SubtypePeripheral blasts (%)Bone marrow blasts (%)Additional featuresAML transformation (%)RARefractory anaemia≤1<510–20RARSRefractory anaemia with ringed sideroblasts>15% ringed sideroblasts in bone marrow10–35RAEBRefractory anaemia with excess blasts5–20>50RAEB-TRefractory anaemia with excess blasts in transformation≥521–29optional Auer-rods60–100CMMLChronic myelomonocytic leukaemia≤20Peripheral monocytosis (>103/µl)>40Bennett JM, et al. Br J Haematol 1982;51:189–199.9
10 Survival by cytogenetic presentation in MDS patients 100908070605040302010Patientsn(%)−Y17(2)del(5q)48(6)Normal489(69)del(20q)16Misc. single74(9)+838(5)Double29(3)Misc. double14Chrom 7 abn.10(1)Misc. complex15Complex66(8)Time (years)123456789101112131415161718Greenberg P, et al. Blood. 1998;89:
11 Risk stratification and prognosis scoring for MDS: IPSS IPSS scoreVariable0.51.01.52.0BM blasts (%)< 55–10–11–2021–30KaryotypeGoodInt.PoorCytopenia(s)0/12/3Influence of karyotype according to IPSSGood = normal, −Y, del(5q), del(20q)Poor = complex (≥ 3 abnormalities) or chromosome 7 anomaliesInt. = all other abnormalitiesGreenberg P, et al. Blood. 1998;89:11
12 Cumulative survival of MDS patients by IPSS AML Evolution102030405060708090100102030405060708090100LowInt-1Int-2HighLowInt-1Int-2HighPercentPercent2468101214161824681012141618YearsYearsGreenberg P, et al. Blood 1997;89:2079–2088.
13 MDS: WHO classification 2008 Blast percentageMDS SubtypeDysplasiaPeripheral blasts (%)Bone marrow blasts (%)Ringed sideroblasts (%)Cytogenetics5q− syndromeMostly DysE< 1%< 5%< 15%5q− soleRA, RN, RT, RCUDDysE, N, TVariousRARS> 15%RCMD2–3 lineagesrareRAEB-11–3 lineages5–9%RAEB-25–19%Auer rods +/-10–19%MDS-U1 lineageBM = bone marrow; DysE = dyserythropoiesis; MDS-U = myelodysplastic syndrome, unclassified; N = neutropenia; pB = peripheral blood; RCMD = refractory cytopenia with multilineage dysplasia; RCUD = refractory cytopenia with unilineage dysplasia; RN = refractory neutropenia; RT = refractory thrombocytopenia; T = thrombocytopenia.Swerdlow SH, et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008:
14 Survival of MDS patients according to transfusion dependency 0.00.10.20.220.127.116.11.18.104.22.168Transfusion-independent patientsProportion survivingTransfusion-dependent patients20406080100120140Time (months)Cazzola M, Malcovati L. N Engl J Med. 2005;352:536-8.14
15 The WHO classification-based prognostic scoring system (WPSS) for MDS Points123WHO subtypeRA, RARS, del(5q)RCMD, RCMD-RSRAEB-1RAEB-2Transfusion requirementNoneRegular–Cytogenetic categoryGoodInt.PoorRisk groupsScoreMedian survival (months)Very low103Low172Intermediate240High3–421Very high5–612Transfusion dependence is an independent indicator of severity of disease and has a significant effect on survivalMalcovati L, et al. J Clin Oncol. 2007;25:15
16 Overall survival and AML risk assessments in MDS by WPSS A. Overall survival at diagnosis (n=426)B. Risk of AML at diagnosis (n=426)C. Time-dependent overall survival (n=271)D. Time-dependent risk of AML (n=271)1.00.90.80.22.214.171.124.30.20.1Cumulative probability of survivalCumulative riskTime (months)Risk groupVery Low Low IntermediateHighVery HighMalcovati L et al. J Clin Oncol 2007;25:3503–3510.16
17 WHO prognosis scoring system allows time dependent prognosis scoring IPSSWPSSProsMore widely used and recognizedAllows time-dependent prognosis scoring and risk stratificationTakes into account individual patient transfusion needConsApplies only at the time of diagnosisUnderestimates the impact of transfusion requirement and cytogeneticsUnderestimates the impact of poor cytogeneticsGreenberg P, et al. Blood 1997;89:2079–2088. Malcovati L, et al. J Clin Oncol. 2007;25:3503–3510.Schanz J, et al. Blood. 2007;110:[abstract 248]. Kantarjian H, et al. Cancer. 2008;113:Garcia-Manero G, et al. Leukemia. 2008;22:
18 Practical guide to bone marrow aspirate analysis in MDS
19 Bone marrow biopsy How is it done: Marrow aspirate and bone (trephine) biopsy are removed by physician in an outpatient procedure under local anesthesiaheparinized sample can be stored at room temperature for 24 hoursWhat tests are done?assessment of cellularity, architecture and focal collection of blasts for hematopoietic dysplasiasmear staining examinationcytogenetic analysiskaryotypeFISHWhat other tests might be done?flow cytometrycell countscell sortingimmunophenotypingHellström-Lindberg E. Myelodysplastic Syndromes. London: Remedica; Van de Loosdrecht, et al, Haematologica.2009; 94: Image from: Medline Plus. NIH/NLM. Accessed Jan, 2011.
20 Analysis of bone marrow smears Bone marrow aspirate smears are prepared on a slide by a medical technician using:Wright’s stainPerl’s staining (for ringed sideroblasts)May-Grünwald-Giemsa stainingImmunohistochemical stainings, i.e. CD34A pathologist or hematologist then examines the slides for cell abnormalities under microscope:at least 400 nucleated cells and 20 megakaryocytes should be examined for morphologyHellström-Lindberg E. Myelodysplastic Syndromes. London: Remedica; 2008.
21 Analysis of bone marrow smears (cont.) Refractory anaemia with excess blasts (RAEB)Refractory anaemia (RA)Abnormal large megakaryocyte (double arrow), abnormal hypo-granular and Pelger neutrophils (single arrows).Erythroid cells with peri-nuclear iron accumulation Prussian blue staining (left); Perl’s stain showing ringed sideroblasts with peri-nuclear (mitochondrial) deposition of iron (right)ASH Image Bank, used with permission, all rights reserved.Courtesy of J. Goasguen, Université de Rennes, France.
22 Immunophenotyping by flow cytometry Various lineages are labeled with antibodies that recognize specific haematopoeitic identifiersCombination of no more than four labels recommendedImportant for identification of blasts - CD34+/abnormal granularity/CD45dimWell-correlated with other diagnostic techniques and prognostic systemsVan de Loosdrecht, et al. Haematologica.2009; 94:Image from:
24 Importance of cytogenetic analysis in MDS Nearly half of the patients with MDS present with cytogenetic abnormalities1changes have a pathogenetic relevance (i.e. loss or gain of gene function)Cytogenetic analysis is essential for the diagnosis and classification of MDS according to IPSS and WPSS2Chromosomal aberrations have prognostic relevance for OS and for the time to leukaemic transformation, independent of other factorsCytogenetic analysis forms the basis for therapeutic decisionscytogenetics is indicative of response to therapy, i.e. lenalidomide in del(5q)3 and azacitidine in −7/del(7q)4IPSS = International Prognostic Scoring System; OS = overall survival; WPSS = WHO classification-based Prognostic Scoring System.1Haase D, et al. Blood. 2007;110: Greenberg P, et al. Blood. 1998;89: List AF, et al. N Engl J Med. 2006;355: Fenaux P, et al. Lancet Oncol. 2009;10:2424
25 Cytogenetic aberrations are frequent in patients with MDS 9%14%29%48%Normal karyotypeOne abnormalityTwo abnormalitiesComplex karyotypeN = 2,072 patients with MDSHaase D, et al. Blood. 2007;110:
26 WHO 2008: Incidence of the most common cytogenetic aberrations in MDS (over 5%) Unbalanced aberrationsDe novo MDS (%)Secondary MDS (%)+810−7/del(7q)50−5/del(5q)40del(20q)5–8−Y5iso(17q)/t(17p)/del(17p)3–5Unbalanced aberrations with loss of genetic information (deletions or monosomies) are most common in MDS; balanced aberrations are rareSwerdlow SH, et al. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC; 2008:441.
27 When should cytogenetic testing be performed in patients with MDS: Diagnosis WHO 2008 guidelines recommend a complete cytogenetic analysis of BM at initial diagnosis in all patients with MDSCytogenetic analysis is mandatory fordiagnosis of MDS associated with del(5q)patients with refractory cytopenia(s) who lack MDS diagnostic features; these patients may be considered as having presumptive evidence of MDS if they have MDS-related cytogenetic abnormalities (slides 4 and 5)rather than indicating abnormality, isolated loss of Y chromosome might be an age-related phenomenon and mosaicism with trisomy 8 might be a constitutional change. Therefore, they might not be sufficient to prove MDSBM = bone marrow.Swerdlow SH, et al. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC; 2008:441. Valent P, Horny HP. Eur J Clin Invest. 2009;39: Vardiman JW, et al. Blood. 2009;114:
28 FAQs: Cytogenetic testing for diagnosis of patients with MDS Is cytogenetic testing required for all patients?Yes, cytogenetic testing should be performed whenever possible at initial diagnosis and every 6–12 months during follow-up since karyotype and prognosis might change during the course of the disease. There are a few exceptions that will have no therapeutic consequences, e.g. very frail and multi-morbid patients.Are there disease presentations that correlate with specific cytogenetic abnormalities?There is no correlation between specific abnormalities and disease presentation (with exception of the association between isolated del(5q) syndrome and RA, with typical dysplasia of megakaryocytes).Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
29 FAQs: Cytogenetic testing during treatment in patients with MDS Why is it important to perform cytogenetic testing during the treatment course?Cytogenetic remissions represent a better quality of remission and are more reliable than those determined by blood films or cytomorphology from the BM. Furthermore, karyotype might change during the course of the disease affecting the prognosis and the treatment of the patient. First data show that a cytogenetic progression might be detectable several weeks before clinical manifestation.Is cytogenetic testing important for all patients or is it recommended for specific focus groups?All patients with clonal abnormalities identified before start of therapy should be followed up during therapy. Also, patients with initially normal karyotype might develop abnormalities during the course of disease and, therefore, require regular monitoring.Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
30 FAQs: Cytogenetic testing during treatment in patients with MDS (cont How often should cytogenetic testing be performed?Cytogenetics from BM is recommended when possible at least once every 6 months during therapy. When FISH is performed using peripheral blood (i.e. CD34-FISH), testing should be conducted every 3rd month.What is the definition of cytogenetic progression in MDS?1) Occurrence of new cytogenetic abnormalities (first in patients with normal karyotype or additional for patients with abnormalities).2) Significant increase (> 50%) of the size of the clone with certain cytogenetic abnormalities.When should treatment be altered as a result of changes in the cytogenetic profile?When a clear cytogenetic progression is seen, or when an abnormal clone is completely eliminated and karyotype turns normal and stays normal over time (at least 3 months); this depends on the type of therapy.Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
31 Chromosome analysis in MDS: Karyotyping Each cell in the body contains 46 chromosomes representing the normal human karyotypeMDS patients frequently have a clonal abnormality in the hematopoietic progenitor cells, where a proportion of these have an abnormal karyotype with altered number of chromosomes and/or large alterations in their structureChanges in the number of the chromosomes is due to monosomies (loss of a chromosome) and/or polysomies (more then 2 copies of a chromosome)Changes in the structure of the chromosomes are commonly noted as:deletions, when part or entire chromatid is missinginsertions, when additional material is included in a chromosometranslocations, when genetic material is exchanged between chromosomes
32 How to perform cytogenetic testing in patients with MDS: Karyotyping SampleCollect at least 10–20 mL of heparinized BM aspiratesStorageSamples should reach the lab within 24 hours after biopsyMetaphase qualityTo detect structural abnormalities, adequate chromosome banding is required (≥ 150–250 bands per karyogram)Number of metaphases neededAnalyse 25 metaphases if possible, especially if the karyotype is normal, to exclude a small aberrant cell cloneSource of the BM aspirate photo:Source of cytogenetics lab photo:Source of metaphase quality photo: Dr D. Haase slidesSource of metaphase photo:678910See presenter for references.
33 How to perform cytogenetic testing in patients with MDS: Karyotyping (cont.) Medical technician prepares metaphase slides for karyotyping, by:culturing bone marrow aspirate for hours, and thenexposing the cells to slightly hypotonic solutionsynchronizing them in metaphase (e.g. using colchicine)finally, staining them (e.g. DAPI staining) and fixing them on slidesA pathologist or hematologist then examines the slides for chromosomal abnormalities under microscope:Samples are examined under microscope manually or with aid of analysis software (separating, enhancing banding pattern, chromosome pairing)At least 25 metaphases should be examined, especially if the karyotype is normal, to exclude a small aberrant cell cloneHolland and Frei. Cancer Medicine 6. American Cancer Society; 2003.Lucia Cytogenetics. Accessed Feb.2011.
34 FAQs: Karyotyping Can peripheral blood be used? Peripheral blood is usually not an alternative. No BM available: attempt banding analysis from peripheral blood (CD34-FISH), especially when blasts are present. Metaphase yield from peripheral blood is generally worse than that from BM aspirates. CD34-FISH can be an option.1What sample-related factors could influence the accuracy of the test?Ex-vivo time over 24 hours: clotting; bacterial/fungal contamination; and low cellular content (e.g. hypocellular BM, or if several syringes are filled during biopsy and the last syringe is used for banding analysis).What are the limitations of this method?Need for dividing cells; cell clones < 10% of abnormal cells can be overlooked; submicroscopic abnormalities cannot be detected.FISH = fluorescent in situ hybridization.1Braulke F, et al. Leuk Res. 2010;34:Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
35 Chromosome analysis in MDS: Fluorescence in situ hybridization (FISH) Fluorescently labeled DNA probes recognize complementary sequences on the chromosomesprobes that recognize centromeres detect changes in the number of chromosomesprobes that recognize specific genes can detect changes in the chromosome sequenceFluorescently labeled probes for bcr and ablgenes show exchange of DNA betweenChromosomes in chronic myelogenous leukemiaImages from: Accessed Feb.2011.
36 Preparation of the FISH sample Metaphase slides for karyotyping could also use bone marrow smears and paraffin-embedded bone marrow biopsies)Denature DNA (heat up the sample)Hybridize with denatured (pre-heated) labeled probesWash to remove excess probeDAPI stain for DNAView with fluorescence microscope and take photographsPreparation of the FISH could be done by trained medical technician or clinical geneticist. The analysis of the samples is carried by clinical geneticists and/or hematologists.Image from: Accessed Feb. 2011Protocol from: labs.mmg.pitt.edu/gjoerup/FISH%20protocol%20Vysis.doc. Accessed Feb
37 FISH: Selection of probes MDS FISH panel for initial diagnosis detects the most common aberrations, and typically includes probes for 5q, 7q, #8, 11q, 12p, 13q, 17p, and 20qProbes*ManufacturerWeb siteMultiprobe MDS/AML panelCytocell5/5q, 7/7q, 8cen, 20qGenzyme Genetics5p/q, 7q, 17p13, 20q13Kreatech5p/q, 7cen/q, 8cen, 17p13, 20q13, YcenAbbott Molecular5/5q, 7/7q, 20qMetasystemsSelection of the FISH probes is essential part of the analysis and is typically done by clinical geneticists in cooperation with hematologists*Examples are not representative of the complete spectrum of probes available from each provider.1Cherry AM, et al. Blood. 2010;116:[abstract 2922].
38 FAQs: FISH How is a probe selected? Use the standard MDS FISH panel for initial diagnosis. When the aberration is known/suspected, use additional probes in the region. If disease morphology is suggestive of a certain genotype, one could directly use the respective probe – e.g. if RA with typical dysplasia of megakaryocytes occurs, then use probes for del(5q). Note, however, that one can miss other genetic changes with this approach.How many probes are typically used in a panel?A standard panel consists of 7–8 probes.1 An extended panel can include up to 12 probes.1Cherry AM, et al. Blood. 2010;116:[abstract 2922].Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
39 FAQs: FISH (cont.) What probes should be used for survival prognosis? The available survival data are based on karyotyping (banding) studies. However, in addition to karyotyping, one should use a standard FISH panel: 5p/q, 7cen/q, #8cen, 17p13, 20q13, and Ycen (in males).What are the limitations of FISH?One sees/finds what one is looking for; therefore, one could overlook complex or rare abnormalities (e.g. finding an isolated del(5q) by FISH might give a false good prognosis if it is a part of a complex genotype that has been misidentified).Questions and answers were prepared under the review of Dr. Haase, University of Göttingen, Germany.
41 SummaryWHO 2008 guidelines recommend complete cytogenetic analysis of BM at initial diagnosis of MDSChromosomal aberrations, among other factors, have prognostic relevance on overall survival and time to leukaemic transformationIPSS was first to define cytogenetic risk groups and to show an association with the survival prognosis of the patientimproved understanding of the cytogenetic risk factors provides a better prognosis scoring for the patients with MDSApproximately 50% of MDS patients have abnormal cytogeneticsin addition, it is supposed that many patients with “normal cytogenetics” actually have clonal abnormalities that remain undetected by metaphase cytogenetics
42 Summary (cont.)Cytogenetic analysis is essential for making therapeutic decisions with regard to patients with MDS as it has shown associations with the response to hypomethylating and immunomodulating agentsSequential cytogenetic analysis is recommended to improve clinical management in MDS
47 GLOSSARY LIC = liver iron concentration LVEF = left-ventricular ejection fractionMCA = middle cerebral arteryMDS = Myelodysplastic syndromesMDS-U = myelodysplastic syndrome, unclassifiedMRA = magnetic resonance angiographyMRI = magnetic resonance imagingMV = mean velocity.N = neutropeniaNEX = number of excitationsNIH = National Institute of HealthOS = overall survival
48 GLOSSARY pB = peripheral blood PI = pulsatility index PSV = peak systolic VelocityRA =refractory anemiaRAEB = refractory anemia with excess blastsRAEB -T = refractory anemia with excess blasts in transformationRARS = refractory anemia with ringed sideroblastsRBC = red blood cellsRF = radio-frequencyRCMD = refractory cytopenia with multilineage dysplasiaRCMD-RS = refractory cytopenia with multilineage dysplasia with ringed sideroblastsRCUD = refractory cytopenia with unilineage dysplasia
49 GLOSSARY RN = refractory neutropenia ROI = region of interest RT = refractory thrombocytopeniaSCD = sickle cell diseaseSD = standard deviationSI = signal intensitySIR = signal intensity ratioSF = serum ferritinSNP-a = single-nucleotide polymorphismSQUID = superconducting quantum interface device.STOP = = Stroke Prevention Trial in Sickle Cell AnemiaSTOP II = Optimizing Primary Stroke Prevention in Sickle Cell Anemia
50 GLOSSARY T = thrombocytopenia TAMMV = time-averaged mean of the maximum velocity.TCCS = transcranial colour-coded sonographyTCD = transcranial doppler ultrasonographyTCDI = duplex (imaging TCD)TE = echo timeTR = repetition timeWHO = World Health OrganizationWPSS = WHO classification-based Prognostic Scoring System
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