Megakaryocyte Staining (Code 20804) Notice of Assessment

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1 Megakaryocyte Staining (Code 20804) Notice of Assessment June 2013 DISCLAIMER: This document was originally drafted in French by the Institut national d'ecellence en santé et en services sociau (INESSS), and that version can be consulted at It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation. While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.

2 1 GENERAL INFORMATION 1.1 Requestor: Hôpital Notre-Dame (affiliated with CHUM) 1.2 Application Submitted: August 17, Notice Issued: April 12, 2013 Note: This notice is based on the scientific and commercial information (submitted by the requestor[s]) and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS. 2 TECHNOLOGY, COMPANY, AND LICENCES 2.1 Name of the Technology Immunocytochemical 16 staining of megakaryocytes (Mk) on culture slide (quantitative assay for number of megakaryocytic colonies). Culturing megakaryocyte progenitors is a test included in the Répertoire québécois et système de mesure des procédures de biologie médicale (the Inde) (code 20632). 2.2 Brief Description of the Technology The technology proposed by the requestor (MegaCult TM -C Collagen with Cytokines) was developed by StemCell Technologies (catalogue numbers and 04951). The kit is optimized for quantification of megakaryocyte progenitors CFU-Mk (colony-forming unit-megakaryocytes) in bone marrow, peripheral blood and cord blood cultures. The kit s components include: A serum-free culture medium, to minimize the inhibitory effects of factors such as transforming growth factor-beta (TGF-β) found in sera used in assays for other hematopoietic lineages A collagen solution for optimal growth of Mk progenitors, containing a combination of cytokines that stimulate Mk colony formation: thrombopoietin TPO, IL-3, IL-6, IL-11 and stem cell factor (SCF) The kit features chamber slides that allow incubation, fiation and staining of the cells of the entire culture on the same slide. CFU-Mk are stained with primary antibody anti-gpllb/llla (CD41), a secondary biotinylated antibody, and an alkaline phosphatase avidin-conjugated detection system to amplify the primary signal. The stained colonies are eamined microscopy and categorized as megakaryocytic colonies (CFU-Mk), mied Mk colonies (containing other lineages) and non-mk colonies. The Mk and platelets, which epress GPllb/llla (CD41), show a pink membrane staining and pale blue nuclei if counterstained with Evans Blue. The colonies can then be subdivided by size: small (3 to 20 cells per colony), produced from more mature progenitors; medium (21 to 40 cells by colony); or large 16 Immunocytochemistry is a technique used to detect the presence of a specific protein on living cells by use of an antibody; the technique is called immunohistochemistry when employed for biological tissue. If the protein is inside the cell (and not at its surface), the cells require fiation and permeabilization before immunodetection. 1

3 ( 50 cells per colony), produced from more primitive Mk progenitors (according to the manufacturer s technical manual). 2.3 Company or Developer StemCell Technologies Inc. This company offers several MegaCult-C Medium kits: with cytokines, without cytokines and with lipids. 2.4 Licence(s): not applicable 2.5 Patent, If Any: not applicable 2.6 Approval Status (Health Canada, FDA) The MegaCult kits are not licensed by Health Canada. The technical manual indicates that they are for research use only. Internal validations are needed. On October 3, 2002, the FDA issued import refusal reports for various StemCell Technologies products because of possible contamination (Operational and Administrative System for Import Support or OASIS 17 ). No other details or updates are available about these refusals. 2.7 Weighted Value: (weighted value in the Inde: 187.0) 3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES 3.1 Targeted Patients Patients presenting uneplained thrombocytosis or thrombocytopenia with suspicion of essential thrombocythemia (ET) 3.2 Targeted Diseases This biological test is used to diagnose ET, one of the myeloproliferative disorders. 18 ET is characterized by the proliferation of the megakaryocytic lineage in bone marrow due to a clonal abnormality in pluripotent stem cells. A complete blood count would therefore show an increased concentration of blood platelets (> /L) for more than si consecutive months, according to the 2008 WHO revised diagnostic criteria for ET (Harrison et al., 2010). (Appendi A lists these criteria.) ET is a diagnosis of eclusion because no specific test eists for this disease. The challenge is to distinguish ET from a thrombocytosis secondary or reactive to an underlying disease (e.g., inflammatory disease and iron deficiency) and other myeloproliferative disorders (chronic myelogenous leukemia, polycythemia vera and myeloid splenomegaly). Clinically, ET is asymptomatic in 40% of cases; manifestations are hemorrhagic in 22% of cases and thrombotic in 33% of cases (Andrieu et al., 1997). Patients with an asymptomatic form of ET have a life epectancy similar to that of unaffected individuals. The prognosis for survival in ET is complicated 17 U.S. Food and Drug Administration. Refusal Details as Recorded in OASIS by FDA for Refusal /4/1 (website). Available at 18 Group of four diseases characterized by an overproduction of one or more types of blood cells that may or may not be accompanied by fibrosis in bone marrow. 2

4 by the risk of thrombotic and hemorrhagic manifestations, leukemic transformation, and progression to myelofibrosis (Serraj et al., 2012). 3.3 Number of Patients Targeted The epected volume is 3 patients every 3 months (about 15 patients annually) for the net 3 years. The incidence of ET in Quebec is 1 to 2.5 cases per 100,000 (information submitted by the requestor). 3.4 Medical Specialties Involved (and Other Professions, If Any) Hematology 3.5 Testing Procedure This biological test requires a bone marrow aspiration performed by a hematologist. It is performed eclusively at CHUM, which does not provide this service to other institutions TECHNOLOGICAL BACKGROUND 4.1 Nature of the Diagnostic Technology: Unique 4.2 Brief Description of the Current Technological Contet According to the 2008 WHO revised criteria, a bone marrow aspirate smear stained with Wright- Giemsa or a similar stain is the diagnostic test to confirm a myeloproliferative neoplasia, including ET (as it confirms a medullary hyperplasia of megakaryocytic lineage with cytological dystrophy 20 ) (Vardiman et al., 2009). It also serves to distinguish ET from secondary thrombocytoses and to rule out other myeloproliferative disorders. 4.3 Brief Description of the Advantages Cited for the New Technology The foundations of this technology are not new. It is based on detecting progenitors of the megakaryocytic lineage in the membrane epression of GPIIb/IIIa (CD41) (Hogge et al., 1997). The marker used is specific to the megakaryocytic lineage (Jean-Pierre, 2005). The advantage of the kit is the use of collagen as a gelling agent, which does not require specialized laboratory equipment and avoids eposing cells to high temperatures (StemCell Technologies, 2010). The kit s collagen solution and other components allow optimal growth of the megakaryocyte progenitors. In addition, the use of commercial culture media promotes standardization of the method and improves inter- and intra-laboratory reproducibility (Mondet, 2011). 4.4 Cost of Technology and Options: Not analyzed 19 Centre Hospitalier de l Université de Montréal (CHUM). Laboratories - Megakaryocytic cultures (website). Available at 20 The presence of enlarged megakaryocytes with multilobed nuclei, most often in clusters of three or more cells. 3

5 5 EVIDENCE 5.1 Clinical Relevance Other Tests Replaced: Not applicable Diagnostic or Prognostic Value Three studies used StemCell Technologies MegaCult TM kits to diagnose ET (Escoffre-Barbe et al., 2006; Dobo et al., 2004; Mi et al., 2001). These studies weighed the diagnostic value of an endogenous culture (in the absence of cytokines) of megakaryocyte colonies detected through immunocytochemical staining with anti-cd41 monoclonal antibody (anti-gpiib/iiia) (Escoffre-Barbe et al., 2006; Mi et al., 2001) or through May-Grünwald-Giemsa staining (Dobo et al., 2004). Their analysis is based on comparing results of the megakaryocytic growth with or without cytokines and does not consider the value of immunocytochemical staining. In the Dobo et al. (2004) multi-centre study, the culture was grown from bone marrow and peripheral blood samples on collagen-based semi-solid medium (n=121). The samples were placed in three culture media: (1) MegaCult with lipids and human collagen, without serum or cytokines; (2) control culture medium, supplemented with various cytokines (interleukins IL-3, IL-6, stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and erythropoietin for erythrocytic progenitor growth (burst-forming unit-erythroid or BFU- E)); and (3) control culture medium, with cytokines (interleukins IL-6, IL-3 and thrombopoietin) for megakaryocytic progenitor growth (CFU-Mk). Megakaryocyte staining was done using the conventional May-Grünwald-Giemsa method. According to the authors, the identification of stained CFU-Mk (> 4 megakaryocytes) is as accurate as immunocytochemical labelling with anti-cd41/61. The bone marrow progenitor cultures (medium with collagen, without serum and without cytokines) confirm that the formation of endogenous megakaryocytic and endogenous erythrocytic progenitor colonies is specific for ET (present in 77.8% and 33.3% of the cases, respectively). No endogenous colony was observed among the patients with reactive (secondary) thrombocytoses (RT). In the peripheral blood cultures, endogenous megakaryocytic colonies were found for 87.8% of ET and 53.1% of RT patients, while endogenous erythrocytic colonies were present in 25.7% of ET cultures and were not formed in RT cultures (0%). These results confirm that this method permits a differential diagnosis between ET and RT. In the control groups (with cytokines), no difference was observed between patients with ET and those with RT, whether in bone marrow cell or peripheral blood cultures. These results therefore reflect very good specificity (100%) and sensitivity (80.2%) of the spontaneous culture (without cytokines), with May Grünwald-Giemsa staining, for diagnosing ET on the basis of endogenous megakaryocytic progenitors isolated from bone marrow. Mi et al. (2001) used a serum-free collagen-based semi-solid culture medium without cytokines, and then with various concentrations of thrombopoietin (TPO)to diagnose ET in bone marrow samples (29 patients and 7 healthy controls). Immunocytochemical staining was done using monoclonal antibody anti-cd41 (Immunotech). The results showed a positive culture for 100% of the 11 patients with ET and for none of the 6 patients with RT (p=0.002), confirmed with the group of healthy controls (p=0.0007). The culture was positive for 33% (4/12) of the patients with polycythemia vera (PV). TPO additions at increasing concentrations (0.5 ng/ml, 5 ng/ml, 50 ng/ml) did not allow discrimination among the diagnoses. The authors conclude that an assay using a serum-free collagen-based medium without cytokines allows for a differential diagnosis between ET and RT. 4

6 The third study analyzed whether a megakaryocytic endogenous culture from bone marrow cells could be used to distinguish ET from polycythemia vera (PV). Growth of CFU-Mk in the presence or absence of cytokines (TPO, IL-3, IL-6) was analyzed using a collagen-based medium (MegaCult). After 14 days, the colonies formed (3 or more cells) were visualized through immunostaining (monoclonal antibody anti-cd41 and alkaline phosphatase). Spontaneous CFU-Mk growth was similarly detected in both diagnoses and observed in 95.2% (40/42) of the patients with ET, compared with 94.0% (47/50) of the patients with PV. The authors conclude that the assay is useful for detecting ET and PV, but not for discriminating between them (Escoffre-Barbe et al., 2006). As a final note, the studies make no distinction between the results associated with the culture method and those associated with a method involving the staining of megakaryocytes. No direct comparison between conventional May Grünwald-Giemsa staining and immunocytochemistry was found Therapeutic Value: No specific information available 5.2 Clinical Validity COMPONENT PRESENCE ABSENCE NOT APPLICABLE Sensitivity Specificity Positive predictive value (PPV) Negative predictive value (NPV) Likelihood ratio (LR) Receiver operating characteristics (ROC) curve Accuracy Culturing method: from a bone marrow biopsy of endogenous megakaryocytic/erythrocytic colonies; conventional May-Grünwald-Giemsa staining (Dobo et al., 2004): Sensitivity: 80.2% for diagnosing ET Specificity: 100% for diagnosing ET Culturing method: serum-free, collagen-based, without cytokines, from a bone marrow biopsy; immunostaining with monoclonal antibody anti-cd41 (Immunotech) (Mi et al., 2001): Sensitivity: 100% for diagnosing ET Sensitivity: 33% for diagnosing PV Perfect discrimination between RT and healthy controls 5

7 Culturing method: spontaneous megakaryocytic colony formation from bone marrow biopsy; immunostaining (monoclonal antibody anti-cd41 and alkaline phosphatase) (Escoffre-Barbe et al., 2006): Sensitivity: 95% for diagnosing ET Sensitivity: 94% for diagnosing PV No discrimination between ET and PV 5.3 Analytical (or Technical) Validity COMPONENT PRESENCE ABSENCE NOT APPLICABLE Repeatability Reproducibility Analytical sensitivity Analytical specificity Matri effect Concordance Correlation 5.4 Recommendations for Listing in Other Jurisdictions The United Kingdom practice guideline for diagnosing essential thrombocythemia (Harrison et al., 2010) does not discuss the diagnostic utility of immunocytochemical staining with a megakaryocyte culture and makes no recommendation in that regard. 6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST 6.1 Impact on Material and Human Resources: Not assessed 6.2 Economic Consequences of Introduction Into Québec s Health and Social Services System: Not assessed 6.3 Main Organizational, Ethical, or Other (Social, Legal, Political) Issues: Not assessed 6

8 7 INESSS NOTICE IN BRIEF Megakaryocyte Staining (Code 20804) Status of diagnostic technology Established Innovative Eperimental (for research purposes only) Replacement of technology:, which becomes obsolete INESSS recommendation Keep test in the Inde Remove test from the Inde Reassess test Additional recommendation Draw connection with listing of drugs, if companion test Produce an optimal use guide Identify indicators, when monitoring is required 7

9 APPENDIX A Table A1: World Health Organization Diagnostic Criteria (2008) (Adapted from Tefferi et al., 2009) Major criteria Minor criteria PV ET PMF 1. One of the following polycythemia criteria: a. Hgb > 18.5 g/dl (men) or > 16.5 g/dl (women) b. Hgb > 17 g/dl (men) or > 15 g/dl (women), if associated with a sustained increase of 2 g/dl from baseline that cannot be attributed to correction of iron deficiency c. Hgb or hematocrit greater than the 99th percentile of reference range for age, se, or altitude of residence d. Red cell mass > 25% above mean normal predicted 2. Presence of JAK2V617F mutation or similar mutation under similar consequences 1. Trilineage myeloproliferation 2. Subnormal serum erythropoietin 3. Endogenous erythroid colony growth Diagnosis Requires meeting either both major criteria and 1 minor criterion OR the first major criterion and 2 minor criteria 1. Platelet count /L 2. Megakaryocytic proliferation with large and mature Mk. No or little granulocyte or erythrocytic proliferation 3. Not meeting WHO criteria for CML, PV, PMF, MDS or other myeloid neoplasm 4. Demonstration of JAK2V617F mutation OR another clonal marker OR no evidence of reactive thrombocytosis Requires meeting all 4 major criteria 1. Megakaryocyte proliferation and atypia (small to large Mk with an aberrant nuclear/cytoplasmic ratio and hyperchromatic and irregularly folded nuclei). These atypia accompanied by either: a. Reticulin and/or collagen fibrosis b. Hypercellularity with granulocytic proliferation and often decreased erythrocytic proliferation 2. Not meeting WHO criteria for CML, PV, MDS or other myeloid neoplasm 3. One of the following elements: a. Demonstration of JAK2V617F mutation b. Demonstration of another clonal marker c. No evidence of reactive bone marrow fibrosis 1. Leukoerythroblastosis 2. Increased serum LDH 3. Anemia 4. Palpable splenomegaly Requires meeting all 3 major criteria and 2 minor criteria Abbreviations: Hgb = hemoglobin; LDH = lactate dehydrogenase; CML = chronic myelogenous leukemia; PMF = primary myelofibrosis; WHO = World Health Organization; PV = polycythemia vera; MDS = myelodysplastic syndrome; ET = essential thrombocythemia 8

10 REFERENCES Andrieu J-M, Colonna P, Lévy R. Cancers : guide pratique d évaluation, de traitement et de surveillance. Paris, France : ESTEM; Dobo I, Boiret N, Lippert E, Girodon F, Mossuz P, Donnard M, et al. A standardized endogenous megakaryocytic erythroid colony assay for the diagnosis of essential thrombocythemia. Haematologica 2004;89(10): Escoffre-Barbe M, Amiot L, Beaucournu P, Jego P, Grulois I, Grosbois B, et al. Spontaneous megakaryocytic colony formation does not discriminate between essential thrombocythemia and polycythemia vera. Am J Hematol 2006;81(7): Harrison CN, Bareford D, Butt N, Campbell P, Conneally E, Drummond M, et al. Guideline for investigation and management of adults and children presenting with a thrombocytosis. Br J Haematol 2010;149(3): Hogge D, Fanning S, Bockhold K, Petzer A, Lambie K, Lansdorp P, et al. Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol 1997;96(4): Jeanpierre S. Rôle des molécules de la famille du TGF-b dans la mégacaryopoïèse. Mémoire présenté pour l obtention du diplôme de l École pratique des hautes études. Lyon, France : École pratique des hautes études (EPHE). Available from: pierre_rdv05.pdf. Mondet J. Caractérisation clinique et biologique d une population de l arc alpin présumée atteinte d un néoplasme myéloprolifératif Philadelphie négatif : apport respectif des différents marqueurs biologiques. Thèse présentée pour l obtention du diplôme d État de docteur en pharmacie. Grenoble, France : Université Joseph Fourier, Faculté de pharmacie de Grenoble; Available from: Mi JQ, Blanc-Jouvan F, Wang J, Sotto MF, Cousin F, Castinel A, et al. Endogenous megakaryocytic colony formation and thrombopoietin sensitivity of megakaryocytic progenitor cells are useful to distinguish between essential thrombocythemia and reactive thrombocytosis. J Hematother Stem Cell Res 2001;10(3): Serraj K, Mecili M, Azzouzi H, Ghiura CF, Andres E. Les thrombocytoses : physiopathologie, diagnostic et traitement. Médecine Thérapeutique 2012;18(1): StemCell Technologies. MegaCult -C: Assays for quantitation of human and mouse megakaryocytic progenitors. Version Vancouver, BC : StemCell Technologies Inc.; Available from: Tefferi A, Thiele J, Vardiman JW. The 2008 World Health Organization classification system for myeloproliferative neoplasms: Order out of chaos. Cancer 2009;115(17): Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: Rationale and important changes. Blood 2009;114(5):