Examples of bone marrow biopsies Normal Hypocellular Lymphoma lymphoid nodule Hoffbrand et al Essential Hematology 2001
Aplastic Anemia (AA) Failure of the marrow to produce adequate numbers of blood cells in the absence of malignancy or dysplasia. Patients have pancytopenia [anemia, neutropenia, & thrombocytopenia] AND a hypocellular marrow. Disease severity: Non-severe: Does not meet criteria for severe Severe: Has 2 or 3 of the following: RBC:retic < 60,000/ l Neut:< 500/ l Plt:<20,000/ l Very Severe: Neut:< 200/ l Bimodal age distribution: teens/20’s & > 60
1998 1999 Aplastic anemia and telomere length…..
Calado, R. T. et al. Blood 2008;111:4446-4455 The chromosome end & the telomerase complex TERT enzymatically adds TTAGGG repeats to the telomere 3’ end using TERC as a template. Dyskerin NOP10, NHP2, and GAR also bind TERC to stabilize the complex.
Telomerase complex mutations and human disease Green: Acquired Aplastic Anemia Red: Dyskeratosis Congenita Black: Pulmonary fibrosis Blue: Polymorphisms Calado, R. T. et al. Blood 2008;111:4446-4455
MPL is the thrombopoietin (TPO) receptor Homodimeric; no other known ligand mpl -/- mice implicate a critical role of TPO/MPL in HSC biology: up to a 25-fold reduction in HSCs! Inactivating Mpl mutations = -Congenital Amegakaryocytic Thrombocytopenia (CAMT) -Progressive aplastic anemia and death very early in life mpl -/- mice live a full life without cytopenias except for platelets. The phenotypic difference between CAMT patients and mpl -/- mice suggests that mpl -/- mice may be a sub-optimal model for studying the role of Mpl in human hematopoietic stem cell biology. TPO/MPL Signaling and Human Hematopoietic Development: MPL protein & gene exon structure : nonsense mutation : missense mutation S : splice site mutation : frame shift deletion
Lymphoma – A Disease of Lymphoid Tissues Non Hodgkin vs Hodgkin (7:1) NHL - ~95% are B cell Continuous spread (HD) vs Hematogenous spread (NHL) The pathologist is everything for making Dx, which in turn, dictates therapy. Indolent (not curable) to aggressive (curable)
Small round lymphocytes Lymphoblast Plasma cells B-Cell Maturation B-cell NHLs correspond in morphology and immunophenotype to normal developing B-cells Large non- cleaved lymphocytes (centroblasts) Small cleaved lymphocytes (centrocytes) Small non- cleaved lymphocytes Memory B-cells Lymphoblastic lymphoma Small lymphocytic lymphoma Mantle cell lymphoma Marginal zone lymphoma Diffuse large B-cell lymphoma* Follicular lymphoma Burkitt lymphoma *DLBCL can likely arise from other stages of B-cell diff. TdT CD5, CD23 CD5 CD10, BCL6 None of these! P. Treseler, UCSF
Small CellsIntermediate CellsLarge Cells Small round Lymphoblast Large non-cleaved Small cleaved Histiocyte Immunoblast Small non-cleaved Small lymphocytic lymphoma Follicular lymphoma Mantle cell lymphoma Marginal zone lymphoma Lymphoblastic Lymphoma Burkitt Lymphoma Diffuse Large B-Cell Lymphoma Low Grade (Indolent) High Grade (Aggressive) Same disease as ALL! Same disease as CLL! P. Treseler, UCSF
Small lymphocytic lymphoma (CLL/SLL) Mantle cell lymphoma (MCL) Follicular lymphoma (FL) Marginal Zone Lymphoma (MZL) Burkitt lymphoma Lymphoblastic lymphoma Diffuse large B-cell lymphoma B-Cell Non-Hodgkin Lymphomas with characteristic translocations t(11;14) t(14;18) t(8;14) P. Treseler, UCSF
Clinical Uses of Hematopoietic Stem Cells I. History/Background for HSC transplant II. Disorders treated III. Types of HSC transplants IV. Source of cells for HSC transplant V. HLA matching: rejection and more – the immunology of BMT VI. Gene Therapy VII. hESCs - A new HSC source? VIII. Some pressing questions in the field
History of Hematopoietic Stem Cell Transplantation A report in 1950 that included classified studies performed in early 1940s by the atomic energy commission.
History of Hematopoietic Stem Cell Transplantation J Lab Clin Med 34:1538, 1949 Surgically mobilized spleens are blocked from irradiation by lead blocks
Bone Marrow Protects Against Radiation Injury J Natl Cancer Inst. 12:197, 1951 Are the protective effects cellular or humoral?
Evidence Favoring the Cellular Theory of BM Protection J Natl Cancer Inst. 15:1023, 1955 DBA/2JN mouse [dark] with BALB/cAnN skin graft [white] after administration of high dose radiation (800 r) and DBA/2JN x BALB/cAnN F 1 bone marrow. Image at 150 days after skin grafting
Hematopoietic Stem Cell Transplantation - The Start of Human Studies From E. Donnall Thomas A Hx of BMT Mid 1950s: realized that BM transplant could be used to treat diseases of the marrow and not just to provide radiation protection Mid 1950s - mid 1960s: Lots of failed attempts for allo transplant -- interest drops 1968: First successful transplant to treat an inherited immunodeficiency using HLA matched sibling 1975: NEJM paper by Thomas* et al reviewed the state of BMT, and helped define critical issues in the field, including HLA matching, infectious complications, GVHD, etc. and showed clearly that one could use this therapy to cure people with leukemia. 1977: Thomas et al report on 100 patients with end stage leukemia treated with HSC transplantation, with 13 long term survivors. 1979: Better results if treat earlier, i.e., in first remission. *E. Donnall Thomas awarded the Nobel Prize in 1990 for BMT
Appelbaum NEJM 2007 Timeline History for Blood and Marrow Transplantation 2008 ~50-60,000/yr worldwide
Transplant activity in the U.S.: 1980-2009 Transplants '80'81'82'83'84'85'86'87'88'89'90'91'92'93'94'95'96'97'98'99'00'01'02'03'04'05'06'07'08'09 Autologous Related Donor Unrelated Donor 10,000 8,000 18,000 12,000 6,000 4,000 2,000 0 16,000 14,000 Year
Indications for hematopoietic stem cell transplant in North America - 2008 3,000 5,500 2,000 1,500 500 0 1,000 2,500 3,500 4,000 4,500 5,000 Number of Transplants Multiple Myeloma NHLAMLHDALLMDS/MPDAplastic Anemia CMLOther Leuk Other Cancer Non- Malig Disease Allogeneic (Total N=6,672) Autologous (Total N=10,302)
Number of allogeneic transplants, by disease, registered with CIBMTR - 1998-2008 Transplants 2,000 3,000 1,000 500 0 1,500 2,500 1999 200020012002200320042005200720082006 * * Data incomplete AML ALL CML AA LYM / MM / CLL Year
1. Autologous - BM (>1,000 ml) or PBSC (~300 ml) 2. Allogeneic - BM or PBSC: Related Unrelated - NMDP 3. Umbilical Cord (<100 ml) Hematopoietic Stem Cell Transplantation: Type & Stem Cell Source
The Hematopoietic Stem Cell Circulation Peripheral Circulation *NOTE: This just shows the endosteal niche for simplicity, but there is also a perivascular niche. Wilson & Trumpp 2006 Circulate Home Lodge Retain Mobilize/Release
HSC sources for clinical application - Bone Marrow 1.General anesthesia in operating room 2. 200- 300 entries into posterior iliac crest yielding >1,000 ml iliac crest yielding >1,000 ml 3.Overnight stay (or more) 4.Sore for a while 5.Net loss of a couple units of blood 6.Dose: > 2 x 10 8 nucleated cells/Kg
HSC sources for clinical application: Peripheral Blood Stem Cells 1. Mobilization - G-CSF [CXCR-4 antagonist] [CXCR-4 antagonist] 2.Two IVs & a cell separator 3.3-4 hours 4.Cytokine can give flu-like symptoms 5.Product is ~250-300 ml 6.“Dose”: 2-5 x 10 6 CD34 + cells/Kg
HSC sources for clinical application: Cord Blood 1.No pain 2.No cytokines 3.<100 ml 4.Dose: >2 x 10 7 nucleated cells 5.Permits greater HLA variation 6.A readily available unrelated source
MHC [Major Histocompatibility Complex] region: encodes antigens that provoke the strongest transplant rejection encodes antigens that provoke the strongest transplant rejection HLA [human leukocyte antigens] genes: The key genes of the MHC cluster, The key genes of the MHC cluster, Located at p21.3 on chromosome 6. Located at p21.3 on chromosome 6. 3 classes - I, II, and III 3 classes - I, II, and III HLA genes are extremely polymorphic and play a key role in T cell activation HLA genes are extremely polymorphic and play a key role in T cell activation associated with the immune response to foreign tissue. associated with the immune response to foreign tissue. Class I: >17 loci Ia = A, B, C, the most important for tissue transplantation Class II: 9 distinct genes; 5 families DR, DQ, DO, DN, & DP Key for HSC transplant: DRB1 and DQB1 FOR HSC Transplant: HLA typing of A, B, C, DR, DQ; 10/10 is the best But clearly more to it than this…. Histocompatibility - Are You a Match?
Currently used drugs to modulate the process Tacrolimus (aka FK-506): binds immunophilin FKBP-12 (FK506 binding protein) creating a new complex, FKBP12-FK506, which interacts with and inhibits calcineurin, thereby inhibiting both T-lymphocyte signal transduction and IL-2 transcription Mycophenolic acid mafatil (MMF): Mycophenolic acid mafatil (MMF): a noncompetitive, selective and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme for nucleotide biosynthesis. Unlike other cell types that can use the salvage biosynthesis pathway, B and T lymphocytes depend on the IMPDH-requiring de novo biosynthesis pathway. Methotrexate: DNA synthesis inhibitor, an anti-folate, and one of the original anti-GVHD drugs. But….some GVHD is good Preventing and treating Graft vs. Host Disease
Annals of Int. Med 1994 Better HLA match does not necessarily mean better outcome Probability of Relapse Probability of Leukemia-free Survival Modern day Allo matching: [10 loci] HLA A, B, C, DR, DQ Graft vs. leukemia provides part of the cure in allogeneic transplants
HSC Transplant for Malignant Disorders: more is not necessarily better Must overcome two opposing immunologic barriers: GVH and HVG 1.Intensive conditioning to: (I) immunoablate & (II) eradicate disease 2.HSC infused to rescue from lethal myeloablation 3.Immuno-modulate recipient (Rx) post transplant SO: Major interest in non-myeloablative conditioning regimens BUT: 1.Myeloablation has lots of toxicity and limits patient age 2.But GVT can be a useful part of GVHD [twins; better outcome w/ some GVHD] 3.Donor lymphocyte infusion (DLI) -best for CML, CLL, and some lymphomas
Gene Marking Studies - The tag sale days “Tag” autologous cells and allowed one to determine if Relapses were from infused cells vs. a failure to eradicate Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Brenner MK, et al. Lancet 31:85, 1993. Pediatric patients with AML; Auto BMT in 1st remission; 2/2 relapses innvoled the ‘tagged’ cells. Genetic marking shows that Ph+ cells present in autologous transplants of CML contribute to relapses after auto BMT in CML. Deisseroth, et al. Blood 83:3068, 1994. But…most relapse is due to failure to eradicate disease in host, and purging autologous samples has not yet proven very useful
Gene Therapy - Correct a Genetic Disease Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease Cavazzana-Calvo M. et al. [Science 288:669, 2000] Sustained Correction of X-linked Severe Combined Immunodefiency by Ex Vivo Gene Therapy Hacein-Bey-Abina S, et al. [NEJM 346:1185, 2002] MLV-based vector, replication- defective, intact LTRs, uses upstream LTR to express c; used retrovirus as supernatant to infect CD34+ cells CD3=T cells X-linked SCID = lack of functional common gamma chain ( c) shared with IL-2, -4, -7, -9, -15, & -21 resulting in failure to develop B, T, and NK cells
Gene Therapy - the bubble pops LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Hacein-Bey-Abina S, et al. [Science 302:415-9, 2003] 9/10 children treated are in remission - great BUT: 2 developed T-cell leukemia with LMO2-linked retroviral insertions - not great 1 additional patient has an LMO2-associated insertion - not great What was known about LMO2: Over-expression in transgenic mice yields T cell leukemias Blocks T cell differentiation in mice Acts as a bridging molecule in transcription factor complexes Expressed in all progenitors and down regulates with differentiation LMO knockout mice fail to develop any hematopoietic lineage Retroviruses integrate into promoters and into actively expressed genes Gene Therapy Insertional Mutagenesis Insights Dave UP, et al. Science 303:333, 2004 & McCormack et al NEJM 350:913-22, 2004
hESCs as a Therapeutic Source of HSCs? Large banks of hESC lines - covers ‘all’ HLA options Manipulation of histocompatibility genes in hESC. Replacement of immune system of patient with hESC-derived HSC prior to transplant. Manipulation of T cell response with antibodies or drugs. Nuclear Transfer involving embryonic stem cells Autologous replacement HSCs using induced pleuripotent stem [iPS] cells
Hematopoietic Stem Cell Transplantation: Areas of Research Expanding HS(P)Cs in vitro for therapeutic use Understanding where the good cells are Purging leukemic/diseased cells from healthy HSCs Lots of immunology: Separating GVHD from GVL effect Better HLA matching Treating GVHD Improved conditioning - Making space - CXCR4 antagonist? Novel sources of HSCs hESCs?