Christian CHABANNON. Centre de Thérapie Cellulaire. Département de Biologie du Cancer & Inserm CBT Institut Paoli-Calmettes, Marseille

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1 Quality controls, new technologies, JACIE requirements Enumeration of HSCs: How do I? session 2015 EBMT Annual Meeting Istanbul, Turkey Saturday, March 21st 2015 Christian CHABANNON Centre de Thérapie Cellulaire. Département de Biologie du Cancer & Inserm CBT Institut Paoli-Calmettes, Marseille

2 Disclosures None relevant to the present presentation

3 Biological relevance of available assays

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5 HOW DO WE DEFINE A HEMATOPOIETIC STEM CELL? Through the use of complex biological assays In vitro or in vivo (transplant experiments) Require complex infrastructures and sophisticated expertise Lack of harmonization Usually low- to medium throughput analyses Most often delayed readout

6 HOW DO WE DEFINE A HEMATOPOIETIC STEM CELL? Through the identification of a complex phenotype Detection of several (multiple) membrane or intracellular antigens No single antigen detection defines a stem cell phenotype CD34+ cell Stem Cell?

7 Weissman and Shizuru, Blood 2008

8 Clinical relevance of available assays

9 CD34+ cell counts Expression of CD34 at the surface of hematopoietic cells defines a subset enriched in stem and progenitor cells Not an homogeneous and pure population of hematopoietic stem cells A subpopulation of true stem cells does not express CD34 (at least in mice)

10 CD34+ cell counts In the context of autologous blood HSCT, there is a long-known relation between the number of infused peripheral blood CD34+ cells and neutrophil and platelet recovery following the administration of high-dose chemotherapy Inverse correlation Requirement for a minimal number of CD34+/kg Is more better? Non linear correlation

11 CD34+ cell counts In the context of allogeneic HSCT, is there a relation between the number of infused donor peripheral blood CD34+ cells and recipient neutrophil and platelet recovery following the administration of a conditioning regimen? Inverse correlation Requirement for a minimal number of CD34+/kg? Is more better? Non linear correlation Does this remain true: For alternative donors (such as haplotype mismatched donors)? With NMA conditioning regimen?

12 CD34+ cell counts In the context of allogeneic HSCT, what is the relevance of CD34+ cell counts for othe types of cell sources? Bone marrow Cord blood

13 CD34+ cell counts Because there is a relation between clinical endpoints after HSCT and the quantity of infused CD34+ cells, and based on the assumption that the number of infused CD34+ cells largely reflects the number of collected cells: CD34+ cell counts on patients or donor blood samples are largely used to guide physicians in their decisions to adjust mobilization treatments and to start / stop aphereses CD34+ cell counts are largely used to monitor cell collection efficiency and characterize the collected cell product in terms hematopoietic potential

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15 Currently available tools for CD34+ cell counts Flow cytometry based assays Several manufacturers sell Equipment Including IT fulfilling recommendations for CD34+ cell identification and adapted to clinical practice Diagnostic kits

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18 Any interest in counting CD34+ cell subsets? CD34+/ CD90-? CD34+ / CD38? CD34+ / CXCR4+? CLINICAL CONTEXT?

19 Are there alternative assays to CD34+ cell counts? Clonogenic assays? Multiple types of assays used in research laboratories Only CFU-GM and BFU-E detection has come to the clinic Delayed readout Automation and image analysis may help reduce intraand inter-laboratory variabiity

20 Are there alternative assays to CD34+ cell counts? Stempredict and HALO assays Are proposed to measure stem cells in cord blood Are calibrated and standardized to ensure accuracy, reliability and reproducibility Incorporate internal proficiency testing every time an assay is performed Use a sensitive ATP bioluminescence readout Produce results within 3, 5 or 7 days so far limited clinical relevance

21 Potency assays? What is Hematopoietic Stem Cell Potency? Stem cell potency is the quantitative and validated measurement of proliferation potential or capacity (biological activity) of the "active" stem cell components of a cord blood, mobilized peripheral blood or bone marrow product, which, when administered to the patient, produces the intended response of engraftment. Stem cell proliferation potential or capacity correlates directly with stem cell self-renewal and primitiveness or "stemness". Thus, the greater the proliferation potential, the greater the self-renewal capacity and the more primitive the stem cell population. The more primitive a stem cell population, the greater its potency. Potency must correlate with a response, but not necessarily the clinical outcome. Thus, stem cell potency does not predict or correlate with "time to engraftment", which is the ability of the engrafted stem cells to produce short-term reconstitution by differentiating and maturing into a specific number of neutrophils, platelets and erythrocytes. HemoGenix, Inc.

22 Potency assays? Potency: The therapeutic activity of a product as indicated by appropriate laboratory tests or adequately developed and controlled clinical data.

23 Are there alternative assays to CD34+ cell counts? Detection of ALDH expressing cells with the ALDEFLUOR assay (ALDECOUNT): identification, evaluation, and isolation of stem and progenitor cells based on their expression of the enzyme aldehyde dehydrogenase (ALDH), rather than cell surface phenotype ALDEFLUOR IS: Optimized for human bone marrow, umbilical cord blood, apheresis, and peripheral blood Used to detect stem and progenitor cells in multiple lineages including hematopoietic, mammary, mesenchymal, endothelial, and neural Adaptable for use with other species and cell types, including cancer stem cells Used to identify only viable cells with an intact cellular membrane Suitable for cryopreserved or fresh samples

24 National, European & international regulations

25 REGULATORY REQUIREMENTS Will apply to three distinct although intimately associated activities that are critical for the success of HSCT Cell collection / Cell procurement Cell engineering / Cell processing / Cell manufacturing Quality controls / Potency assays

26 CRITICAL IMPORTANCE OF RESULTS OF QUALITY CONTROLS AND POTENCY ASSAYS Release and distribution of cell therapy products will be under the responsibility of a «qualified person» who will use all results of laboratory tests performed on the donor and at all steps of cell processing Critical importance of these results

27 REGULATORY REQUIREMENTS FOR DIAGNOSTIC TESTS ISO is now required in several European countries including France for all laboratories performing diagnostic tests Is a cell processing facility performing diagnostic tests? Cell engineering / cell manufacturing: NO In-control process:? Debated? Controls on patients and donors that undergo cell collection: YES Using results to support medical decisions such as change in mobilization treatment, starting / stopping aphereses

28 ISO has gained standing as mandatory accreditation in Australia, the Canadian province of Ontario and some EU countries including France, Belgium for molecular virology and oncology testing, Germany for newborn screening, and Latvia for hospital labs. Swiss labs are subject to fewer inspections when accredited. In Belgium it is the standard by which molecular diagnostic tests are reimbursed.

29 REGULATORY REQUIREMENTS FOR DIAGNOSTIC TESTS US equivalents CAP (College of American Pathologists) accreditation programs Clinical Laboratory Improvement Amendments (CLIA) The Centers for Medicare & Medicaid Services (CMS) regulates all laboratory testing (except research) performed on humans in the U.S. through the Clinical Laboratory Improvement Amendments (CLIA) The objective of the CLIA program is to ensure quality laboratory testing.

30 REGULATORY REQUIREMENTS FOR DIAGNOSTIC TESTS Preparing for ISO accreditation is a challenging experience! ISO standards are split in two parts: Chapter 4 Chapter 5 Selecting adopted rather than adapted methods somewhat alleviates your burden. An opportunity to share your pain with your colleagues! Assays for CD34+ cell counts fall in the immunohematology domain

31 Chapter 4 of ISO Standards Technical requirements Qualification of personnel All categories of personnel are concerned Initial training / qualification Habilitation / re-habilitation Maintenance & Continuous control of practical and theoretical knowledge Technical, ethical, QM aspects

32 Potency assays? Definition? For ATMPs, the wording is that «the assay defines the product» Since hematopoietic grafts prepared for «standard» (historical forms of?) autologous or HSCT do not (yet?!) qualify as ATMPs, there is so far no requirement to incorporate nor define a potency assay for our most widely used practices

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34 FACT-JACIE requirements

35 FACT-JACIE Standards 6th Edition recently released Global structure of the standards has remained unchanged Section B: Clinical Sections CM & C: Collection Section D: Processing Basic principle: JACIE requirements do not supersede national, European or international regulations

36 FACT-JACIE Standards There is no specific section in FACT-JACIE standards dedicated to laboratory controls Hovever biological assays are critical tools to fulfill many JACIE requirements

37 Immunogenetics Exception is laboratory in charge of HLA-typing and related biological testing (determination of anti-hla antibodies) Must be EFI or ASHI or equivalent accredited

38 New Standards in section D Quality controls on cryopreserved cellular therapy products D9.2.2 There shall be a written stability program that evaluates the viability and potency of cryopreserved cellular therapy products, minimally annually.

39 New Standards in section D Qualification plans / Validation D Qualification plans shall be reviewed and approved by the Processing Facility Director or designee. D Each validation shall include: D An approved validation plan, including conditions to be validated. D Acceptance criteria. D Data collection. D Evaluation of data. D Summary of results.

40 Qualification plans / Validation Same applies to collection Cell separators used for aphereses C4.13 The Quality Management Plan shall include, or summarize and reference, policies and procedures for qualification of critical reagents, supplies, equipment, and facilities. C Qualification plans shall be reviewed and approved by the Apheresis Collection Facility Director or designee.

41 C4.14 The Quality Management Plan shall include, or summarize and reference, policies and procedures for validation and/or verification of critical procedures to achieve the expected end-points, including viability of cells and cellular therapy product characteristics. C Critical procedures shall include at least the following: collection procedures, labelling, storage, and distribution. C Each validation shall include: C An approved validation plan, including conditions to be validated. C Acceptance criteria. C Data collection. C Evaluation of data. C Summary of results.

42 ACKNOWLEDGEMENTS Claude Lemarié Guillaume Bouchet Jerôme Couquiaud Charlotte Durousseau Julie Gaudée Pierre Lignée Boudra Makhni Sarah Ouffaï Yannis Ratouchniak Lionel Regimbaud Isabelle Sielleur Olivier Vicari Carine Malenfant Didier Bechlian Maelys Berthomieu Patricia Parc Sylvie Portelli Martine Bouyssie Nathalie Scarella Boris Calmels Eric Bertrand Fabien Durand