Hematopoiesis: Part II Abnormal hematopoiesis: -Malignant – Leukemia & Myelodysplasia -Failure – Aplastic Anemia -Lymphoma Clinical Uses of Hematopoietic Stem Cells The Hematopoietic Development Tree & Disease eosinophil Normal MYELOID (CMP) Acute Leukemia LTHSC STHSC neutrophil GMP monocyte basophil MPP MEP erythrocytes platelets Chronic Leukemia megakaryocyte LYMPHOID (CLP) Stem Cells Multipotent Progenitor Cells Committed Precursor Cells T T lymphocyte B B lymphocyte Mature Cells Leukemia Classification Acute (>20% blasts in marrow) Chronic Lymphoid Acute Lymphoblastic Leukemia Precursor B-cell ALL Precursor T-cell ALL Rapid progression Chronic Lymphoid Leukemias Chronic Lymphocytic Leukemia Prolymphocytic Leukemia Slow progression Hairy Cell Leukemia Multiple Myeloma Waldenstrom’s Macroglobulinemia Myeloid Acute myeloid leukemia (AML) • AML recurrent cytogenetic abnorm •AML, minimally differentiated Arrested maturation •AML, without maturation •AML, with maturation •Acute promyelocytic leukemia •Acute myelomonocytic leukemia •Acute monoblastic leukemia •Acute erythroid leukemia Myeloproliferative Disorders Chronic myeloid leukemia Polycythemia vera Essential Thrombocythemia Full maturation…. Chronic idiopathic myelofibrosis just too much of everything Myelodysplastic Syndromes Refractory anemia Refractory anemia with RS Refractory anemia with excess blasts Chronic myelomonocytic leukemia •Acute megakaryoblastic leukemia Acute Leukemias (mature/maturing cells) Lymphoid (immature cells) Chronic Leukemias CLL Myeloid ALL AML CML Leukemia Classification Acute Chronic Lymphoid Acute Lymphoblastic Leukemia • Precursor B-cell ALL • Precursor T-cell ALL Chronic Lymphoid Leukemia • Chronic Lymphocytic Leukemia • Prolymphocytic Leukemia • Hairy Cell Leukemia • Multiple Myeloma • Waldenstrom’s Macroglobulinemia Myeloid Acute myeloid leukemia (AML) • AML recurrent cytogen abnorm •AML, minimally differentiated •AML, without maturation •AML, with maturation •Acute promyelocytic leukemia •Acute myelomonocytic leukemia •Acute monoblastic leukemia •Acute erythroid leukemia Myeloproliferative Disorders • Chronic myeloid leukemia (CML) • Polycythemia vera (PV) • Essential Thrombocythemia (ET) • Chronic idiopathic myelofibrosis (MF) Myelodysplastic Syndromes • Refractory anemia • Refractory anemia with RS • Refractory anemia with excess blasts • Chronic myelomonocytic leukemia •Acute megakaryoblastic leukemia Myelodysplastic Syndromes - myelodysplasia WHO Classification 2008 Myelodysplastic Syndromes - myelodysplasia Prognostication Examples of bone marrow biopsies Normal Lymphoma Hypocellular 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/ml Neut: < 500/ml Plt: <20,000/ml Very Severe: Neut: < 200/ml • Bimodal age distribution: teens/20’s & > 60 Aplastic Anemia Table 1. A Classification of Aplastic Anemia: Acquired (80%): Primary: Idiopathic – most often auto-immune Secondary: Ionizing radiation – accidental exposure Chemicals: benzene, DTT, organophosphates, ecstasy Rx: chemotherapy agents, psychotropic agents, anticonvulsants, NSAIDs, chloramphenicol Viruses: hepatitis – usually nonA, nonB, nonC, nonG EBV CMV [in immunosuppressed host] Paroxysmal Nocturnal Hemoglobinuria (PNH) Congenital (20%): Fanconi’s anemia Dyskeratosis Congenita Amegakaryocytic Thrombocytopenia Other rare disorders Aplastic anemia and telomere length….. 1998 1999 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. Calado, R. T. et al. Blood 2008;111:4446-4455 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 Clinical Consequences of Telomere Erosion Dyskeratosis Congenita Nail dystrophy Hypo pigmentation Marrow Aplasia Pulmonary Fibrosis Oral Leukoplakia Leukemia & dysplasia Hepatic Fibrosis TPO/MPL Signaling and Human Hematopoietic Development: • MPL is the thrombopoietin (TPO) receptor • Homodimeric; no other known ligand • mpl-/- mice implicate a critical role of TPO/MPL in HSC biology: • 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 up to a 25-fold reduction in HSCs! 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 MPL protein & gene exon structure : nonsense mutation hematopoietic stem cell biology. : 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) B-cell NHLs correspond in morphology and B-Cell Maturation immunophenotype to normal developing B-cells Plasma cells Lymphoblast Small round lymphocytes Memory B-cells TdT Lymphoblastic lymphoma CD5 Large noncleaved lymphocytes (centroblasts) Mantle cell lymphoma CD5, CD23 Small lymphocytic lymphoma *DLBCL can likely arise from other stages of B-cell diff. Small noncleaved lymphocytes Small cleaved lymphocytes (centrocytes) CD10, BCL6 Burkitt CD10, BCL6 CD10, BCL6 lymphoma Diffuse large Follicular B-cell lymphoma lymphoma* None of these! Marginal zone lymphoma P. Treseler, UCSF Low Grade (Indolent) Small Cells High Grade (Aggressive) Histiocyte Intermediate Cells Same disease as ALL!Cells Large Lymphoblastic Lymphoma Small round Same disease as CLL! Small cleaved Lymphoblast Large non-cleaved Diffuse Large B-Cell Lymphoma Burkitt Lymphoma Small non-cleaved Immunoblast •Small lymphocytic lymphoma •Follicular lymphoma •Mantle cell lymphoma •Marginal zone lymphoma P. Treseler, UCSF B-Cell Non-Hodgkin Lymphomas with characteristic translocations • • • • • • • Small lymphocytic lymphoma (CLL/SLL) t(11;14) Mantle cell lymphoma (MCL) t(14;18) Follicular lymphoma (FL) Marginal Zone Lymphoma (MZL) t(8;14) Burkitt lymphoma Lymphoblastic lymphoma Diffuse large B-cell lymphoma 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 Surgically mobilized spleens are blocked from irradiation by lead blocks J Lab Clin Med 34:1538, 1949 Bone Marrow Protects Against Radiation Injury Are the protective effects cellular or humoral? J Natl Cancer Inst. 12:197, 1951 Evidence Favoring the Cellular Theory of BM Protection DBA/2JN mouse [dark] with BALB/cAnN skin graft [white] after administration of high dose radiation (800 r) and DBA/2JN x BALB/cAnN F1 bone marrow. Image at 150 days after skin grafting J Natl Cancer Inst. 15:1023, 1955 Hematopoietic Stem Cell Transplantation The Start of Human Studies 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. From E. Donnall Thomas A Hx of BMT 1979: Better results if treat earlier, i.e., in first remission. *E. Donnall Thomas awarded the Nobel Prize in 1990 for BMT Timeline History for Blood and Marrow Transplantation 2008 ~50-60,000/yr worldwide Appelbaum NEJM 2007 Diseases Commonly Treated with HSC Transplantation AUTOLOGOUS* Cancers Multiple Myeloma Non-Hodgkin’s lymphoma Hodgkin’s disease Acute myeloid leukemia Neuroblastoma Ovarian cancer Germ-cell tumors Other Diseases Autoimmune disorders Amyloidosis *>30,000 annually worldwide; 2/3 for lymphoma and multiple myeloma ALLOGENEIC** Cancers Acute myeloid leukemia Acute lymphoblastic leukemia Chronic myeloid leukemia Chronic lymphocytic leukemia Myeloproliferative disorders Lymphoma (Non-HD & HD) Multiple myeloma Myelodysplastic syndromes Other Diseases Aplastic Anemia Sickle cell anemia Thalassemia major Paroxysmal nocturnal hemoglobinuria SCIDs Inborn errors in metabolism **>15,000 annually; >50% for acute leukemias Copelan NEJM 2006 Transplant activity worldwide: 1980-2009 35,000 Autologous 30,000 Allogeneic Transplants 25,000 20,000 15,000 10,000 5,000 0 '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 Year Transplant activity in the U.S.: 1980-2009 18,000 16,000 Transplants 14,000 Autologous Related Donor Unrelated Donor 12,000 10,000 8,000 6,000 4,000 2,000 0 '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 Year Indications for hematopoietic stem cell transplant in North America - 2008 5,500 Allogeneic (Total N=6,672) 5,000 Autologous (Total N=10,302) Number of Transplants 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 Multiple Myeloma NHL AML HD ALL MDS/MPD Aplastic Anemia CML Other Leuk Other Cancer NonMalig Disease Number of allogeneic transplants, by disease, registered with CIBMTR - 1998-2008 3,000 AML ALL CML AA LYM / MM / CLL Transplants 2,500 2,000 1,500 1,000 500 0 1999 2000 2001 2002 2003 2004 Year 2005 2006 2007 * 2008 * * Data incomplete Hematopoietic Stem Cell Transplantation: Type & Stem Cell Source 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 Source for Transplants: Unrelated Donors ~45% ~15% The Hematopoietic Stem Cell Circulation Peripheral Circulation Mobilize/Release Circulate Retain Lodge Home *NOTE: This just shows the endosteal niche for simplicity, but there is also a perivascular niche. Wilson & Trumpp 2006 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 3. Overnight stay (or more) 4. Sore for a while 5. Net loss of a couple units of blood 6. Dose: > 2 x 108 nucleated cells/Kg HSC sources for clinical application: Peripheral Blood Stem Cells 1. Mobilization - G-CSF [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 106 CD34+ cells/Kg HSC sources for clinical application: Cord Blood 1. No pain 2. No cytokines 3. <100 ml 4. Dose: >2 x 107 nucleated cells 5. Permits greater HLA variation 6. A readily available unrelated source Histocompatibility - Are You a Match? MHC [Major Histocompatibility Complex] region: encodes antigens that provoke the strongest transplant rejection HLA [human leukocyte antigens] genes: The key genes of the MHC cluster, Located at p21.3 on chromosome 6. 3 classes - I, II, and III HLA genes are extremely polymorphic and play a key role in T cell activation 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? Unrelated allo donor with 10/10 match Preventing and treating Graft vs. Host Disease 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): 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 Probability of Relapse Probability of Leukemia-free Survival Better HLA match does not necessarily mean better outcome Modern day Allo matching: [10 loci] HLA A, B, C, DR, DQ Graft vs. leukemia provides part of the cure in allogeneic transplants Annals of Int. Med 1994 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 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 SO: Major interest in non-myeloablative conditioning regimens 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] 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 MLV-based vector, replicationdefective, intact LTRs, uses upstream LTR to express c; used retrovirus as supernatant to infect CD34+ cells CD3=T 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? ….and that’s all folks!