Genetics in the management leukemia and lymphoma

Genetics in the Management of Leukemia and Lymphoma Terry Hayes, M.D., Ph.D. Overview • Many subtypes of acute and chronic leukemias are associated with specific changes in the DNA. • These genetic lesions can be used for diagnosis and prognosis. • Understanding the molecular defects may lead to improvement in therapeutic approach. Types of Genetic Lesions in Hematologic Malignancies • Translocations that result in abnormal gene expression due to placement of genes near enhancer or promoter elements of other genes. • Translocations that produce chimeric fusion proteins with biologic activity. • Mutations in known oncogenes/tumor suppressor genes. Consequences of Recurring Chromosomal Translocations Gene A Gene B Altered expression of normal gene B protein Leukemia, Lymphoma A B Expression of fusion proteins Leukemia, Lymphoma, Sarcoma t(8;14) Consequence of Translocation • The immunoglobulin gene causes an abnormal or altered expression of the normal protein produced by the other gene. • It is the abnormal expression, timing and level that are associated with the malignant phenotype. Myelodysplastic Syndromes Myelodysplastic Syndromes • • • • Refractory anemia Refractory anemia with ringed sideroblasts Refractory anemia with excess blasts Refractory anemia with excess blasts in transformation • Chronic myelomonocytic leukemia Genetic Abnormalities in MDS • Found in almost 50% of cases. • Non-random. • Different mutations associated with particular types of MDS. Common Cytogenetic Abnormalities in MDS Abnormality Loss of all or part of chr. 5 Trisomy 8 Loss of all or part of chr. 7 Deletion of 11q, 12p, 20q Deletion of X or Y Incidence 13-27% 5-19% 5-20% 1-7% 2-5% Genes Found on 5q• • • • IL-4 IL-5 GM-CSF M-CSF • PDGF receptor • CD-14 • M-CSF receptor (fms oncogene) 5q- Ringed Sideroblast • 5q-, most common abnormality in MDS • In 27% of MDS • Associated with refractory anemia and refractory anemia with ringed sideroblasts Abnormalities Associated with 5q- Megaloblastic change Abnormal erythroid precursors Abnormalities Associated with 5q- Micromegakaryocyte Pseudo Pelger-Huët Neutrophil Myelodysplastic Syndromes Mutations in ras genes: N-ras, K-ras, H-ras • Found in 10 to 25% of MDS patients • Ras gene mutation most common in CMML Secondary MDS • After exposure to epipodophyllotoxins and anthracyclines • Balanced translocations involving: – 3q26 – 11q23 – 21q22 FISH in MDS 5qMonosomy 7 Trisomy 8 Fluoroscein labeled 5p Rhodamine labeled 5q Red centromere probe Prognostic Subgroups in MDS • Poor – translocations involving chromosomes 1, 3, 6 • Variable – deletions of chromosomes 11q, 13q, 12q, 9q – trisomy 8 • Good – deletions of 5q, 20q; trisomy 21 Papenhausen et al., Cancer Control 1997; 4: 389. Acute and Chronic Leukemias FAB Classification Lymphoid Myeloid M0 l M4 M1 l M5 M2 l M6 M3 l M7 • L1 • L2 • L3 • • • • FAB Classification and Associated Genetic Abnormalities FAB Common Subtype Name M0 Acute myeloblastic leukemia with minimal differentiation (3%) M1 Acute myeloblastic leukemia without maturation (15-20%) M2 Acute myeloblastic leukemia with maturation (25-30%) M3 Acute promyelocytic leukemia (5-10%) Associated Gene Change inv(3q26) and t(3;3) 1% --t (8;21) t(6;9) t(15;17) t(11;17) t(5;17) 40% 1% 98% 1% 1% NEJM 1999:341:1052 FAB Classification and Associated Genetic Abnormalities FAB Common Subtype Name M4 Acute myelomonocytic leukemia (20%) M4eo Acute myelomonocytic leukemia with abnl eosinophils (5-10%) M5 Acute monocytic leukemia (2-9%) M6 M7 Erythroleukemia (3-5%) Acute megakaryocytic leukemia Associated Gene Change, % 11q23 20% inv (3q26), t(3;3) 3% inv(16), t(16;16) 80% 11q23 t(8:16) 20% 2% t(1;22) 5% NEJM 1999:341:1052 Acute Lymphocytic Leukemia • Over 40 known translocations. • Correlate with prognosis. • Can be used to direct therapy. B CELL DIFFERENTIATION Ig Cell Surface Markers CD19 CALLA (CD10) CD20 CD38 Precursor B Cell Leukemias CLL, B Cell Lymphomas Waldenström’s, Myeloma ALL L1 and L2 • Can be B or T cell lineage. • May be hyperdiploid. • Common translocations – t(9;22), t(4;11), t(1;9) Philadelphia Chromosome • t(9;22)(q34;q11) • Found in 90-100% of adult CML 20-30% of adult ALL 5% of childhood ALL • Results in bcr-abl fusion protein – c-abl on chromosome 9 (tyrosine kinase) – bcr on chromosome 22 (function?) Philadelphia Chromosome • In childhood ALL and 50% of adult ALL, bcr exon 1 is fused with c-abl exon 2 = 190 kd. • In CML and the other 50% of adult ALL, bcr exon 2 or 3 is fused to c-abl exon 2 = 210 kd. • Both are active tyrosine kinases. BCR-ABL Protein • Wild-type abl gene shuttles between nucleus and cytoplasm. • Constitutively active tyrosine kinase confined to cytoplasm. • Phosphorylates multiple substrates. • Can transform hematopoietic cells. • Protects hematopoietic cells from apoptosis. BCR-ABL RAS STAT Phosphatidylinositol-3 kinase MYC RAF JUN KINASE AKT STI 571 • • • • Specific Bcr-Abl inhibitor. Daily oral therapy. Given to patients in chronic phase. All patients achieved complete hematologic responses, and some had cytogenetic responses. • Minimal side effects. ALL L1 or L2 • t(4;11)(q21;q23) • Biphenotypic markers • Infants <1 yr. or adults – >90% of infantile ALL • MLL gene translocated to AF-4 • High wbc >150K, worst prognosis ALL L1 or L2 • • • • t(1;19)(q21;p13). One fourth of childhood pre B-cell ALL. E2A and PBX1 are transcription factors. E2A-PBX1 fusion protein combines the transcriptional activation domain of E2A with the DNA binding domain of PBX1. E2A-PBX1 Fusion Protein • Normal E2A gene helps regulate expression of kappa light chain gene. • PBX1 is homologous to the DNA binding region of homeobox genes – homeobox genes regulate the expression of genes critical to embryonic development. ALL L3/Burkitt’s • B cell or Burkitt's type leukemia. • 5% of ALL. • Large round cells with deeply basophilic cytoplasm and vacuoles. ALL - L3 • Associated chromosomal translocations include t(8;14), t(2;8), t(8;22). • Poor prognosis with standard ALL treatment regimens, better with intensified regimens. ALL L3/Burkitt’s Lymphoma IgH Igl Igk Genetic Changes in T-ALL • Transcription factors translocated to T cell receptors in many T-cell ALL – TCR a/d at 14q11-14 – TCR b at 14q34-36 Genotypes in ALL Prognostic Significance in ALL • Favorable prognosis: – hyperdiploid (>50 chromosomes) – t(12;21) • Poor prognosis: – t(9;22) – t(4;11) – t(1;19) Ph Positive ALL • Worst outcome in childhood ALL. • DFS 49% at 5 years if wbc at diagnosis <50,000. • DFS 20% at 5 years if wbc at diagnosis >100,000. • Patients do better with bone marrow transplantation from a HLA-matched donor. NEJM 2000;342:998. Survival in Ph+ ALL Transplantation Chemotherapy Alone NEJM 2000;342:998. Lower Risk • B lineage ALL • Age 1 to 9 • wbc < 50,000 Very High Risk •Ph chromosome • t(4;11) • wbc   •  1 y.o. with MLL AML M2 • t(8;21)(q22;q22) • 7% of AML • Splenomegaly, chloromas • Gene fusion ETO/AML1 AML M2 • More mature leukemic cell • Prominent Auer rods. • Good prognosis with t(8;21) • t(8;21) is 15% of AML AML M2 8;21 Translocation • Involves AML1 gene on chromosome 21 and ETO on chromosome 8. • AML1 is a protein critically involved in binding DNA; regulates transcription of many myeloidspecific genes. Binds with CBFb. • Translocation breaks AML1 in the middle of the gene, removes the transactivation domain and replaces it with part of ETO. AML1-CBFb Complex Other Translocations Involving AML1 • AML1 fused to beta-myosin heavy chain gene in inv(16), seen in AML M4 • AML1 and TEL genes involved in 12;21 translocation seen in 3-% of B progenitor pediatric ALL. • AML1 important in substantial proportion of both AML and ALL. AML M3 • Acute promyelocytic leukemia. • t(15;17). • Fuses PML with retinoic acid receptor. • DIC. • Good prognosis. AML M3 • Fuses PML gene on chromosome 15 with retinoic acid receptor on chromosome 17. • The retinoic acid receptor binds normally. • The PML gene is associated with histone deacetylators, which change the function of genes. • The histone tails wind around the DNA, and the gene is inactivated. AML M3 • Normal physiologic levels of retinoic acid cannot activate the target genes. • Pharmacologic levels of retinoic acid dissociate the histone deacetylation complex and allow the gene to activate, leading to cell differentiation. • Variant of M3 involves 11;17 translocation, with PLZF gene instead of PML. • No response to ATRA. AML M4 • Myelomonoblastic leukemia. • 7-10% of AML. • M4 Eo subtype. AML M4 Eo • • • • Inv(16) or t(16;16). Breakpoint at 16q22. >90% of M4 Eo. Good prognosis. M4 Eo • Involves the CBFb gene (normally bound to AML1, the gene involved in M2 AML). • CBFb gene is also involved in pediatric ALL and many rare translocations in AML; one of the most frequent targets of chromosome rearrangements in leukemia. AML1-CBFb Complex M4 Eo • CBFb subunit is fused to smooth muscle myosin heavy chain gene on chromosome 16. • Sequesters AML1 into functionally inactive complexes within the cytoplasm. AML M4 or M5 • • • • t(11q23;variable). Approximately 5% of M4 and M5 AML. MLL gene. Poor prognosis. MLL Gene • “MLL”: present in myeloid, lymphoid and mixed lineage leukemias. • At least 38 different translocations, most often found in M4 and M5 AML. • 19 of 38 have been cloned. • Leukemias involving MLL lesion generally have a poor prognosis. Location of MLL Translocation Partners AF1p CHROMOSOMES AML ALL other AF1q AF9 AF4 AF5 AF6q21 AF10 AB11 MLL AF6 1 2 3 4 CBP 5 6 7 EEN ENL ELL 8 9 10 11 12 AF15 AF17 MSF CDCrel p300 AFX 13 14 15 16 17 18 19 20 21 22 X Y AML M5 • • • • Monoblastic t(9;11)(p22;q23) Children, young adults Skin or gums infiltrated with leukemic cells • MLL translocated to 9p22 AML M6 • Erythroid leukemia. • 5% of AML. • Often associated with deletions in chromosomes 5 and 7. • Poorer prognosis, often preceded by myelodysplastic syndrome. • Seen in older patients. AML M7 • Megakaryoblastic leukemia • 10% of AML • Trisomy 21, inversions or translocations of chromosome 3, t(9;22); t(1;22) in infants. • Also seen in Klinefelter’s syndrome XXY. Poor Prognostic Features in AML Response to induction chemotherapy: • Unfavorable karyotype • Age >60 yr • Secondary AML • Poor performance status • wbc > 20,000 • Unfavorable immunophenotype Likelihood of relapse: • Unfavorable karyotype • Age >60 yr • Delayed response to induction chemo • wbc > 20,000 • Female sex • Elevated LDH Prognostic Features in AML • t(8;21), t(15;17), and inv(16) have good prognosis. – More frequent in younger patients • Abnormalities of chromosomes 5 or 7, trisomy 8, and t(9;11) have poor prognosis. – More common in older patients and patients with secondary leukemias Overall Survival in AML Favorable Cytogenetic Abnormalities t(8;21) inv (16) t(15;17 Normal cytogenetics Unfavorable Cytogenetic Abnormalities Normal cytogenetics complex -5 del (5q), abnl (3q), -7 Grimwade, Blood 1998;92:2322. Non-Hodgkin’s Lymphoma Burkitt’s Lymphoma • t(8;14)(q24;q32) – c-myc to IgH • t(8;22)(q24;q11) – c-myc to Igl • t(2;8)(p11;q24) – Igk to c-myc • C-myc product regulates gene transcription. Burkitt’s Lymphoma • c-myc breakpoint differs in sporadic and endemic Burkitt’s. • Epstein-Barr Virus: – 97% of endemic Burkitt’s. – 20-30% of sporadic cases. Follicular Lymphomas • t(14;18)(q32;q31). • Present in 85-90% of follicular lymphomas. • bcl-2 on chromosome 18, regulator of apoptosis. • Ig heavy chain on chromosome 14. Mantle Cell Lymphoma • t(11;14)(q13;q32) • bcl-1 or PRAD oncogene encoding cyclin D1 protein on chromosome 11, regulates cell division. • Ig heavy chain region on chromosome 14. T Cell Lymphomas • Translocations often involve T cell receptor genes  a and d chain genes on 14q11-14  b chain gene on 14q34-36  g chain gene on 7p15 • Several other oncogenes – Ttg1, LylI, HOX 11 – Transcription factor families New Directions Spectral karyotyping (SKY) • Identifies all chromosomes simultaneously • Improves precision of karyotype analysis in malignant cells Rowley et al, Blood 1999;93:2038. Summary: Genetic Changes in Leukemia and Lymphoma • Most identified chromosome changes are translocations that place critical hematopoietic genes (immunoglobulin or T cell receptor genes) near other genes that regulate transcription or cell proliferation. • Some translocations produce chimeric fusion proteins with abnormal biologic activity. Summary: Genetic Changes in Leukemia and Lymphoma • Further understanding will allow us to deliver genotype-specific therapy to treat each individual malignancy in the optimal fashion.