Chromosomal Rearrangements in Cancer and Leukemia

Cancers Caused by Chromosomal Rearrangements

• Case 14: CML and ALL

Cancer Statistics (2008 Estimates)

• US Cancer Cases (Excluding skin cancers and in situ carcinomas except urinary bladder)
• Men: 720,280
• Prostate: 33%
• Lung & bronchus: 13%
• Colon & rectum: 10%
• Urinary bladder: 6%
• Melanoma of skin: 5%
• Non-Hodgkin lymphoma: 4%
• Kidney: 3%
• Oral cavity: 3%
• Leukemia: 3%
• Pancreas: 2%
• All Other Sites: 18%
• Women: 679,510
• Breast: 31%
• Lung & bronchus: 12%
• Colon & rectum: 11%
• Uterine corpus: 6%
• Non-Hodgkin lymphoma: 4%
• Melanoma of skin: 4%
• Thyroid: 3%
• Ovary: 3%
• Urinary bladder: 2%
• Pancreas: 2%
• All Other Sites: 22%

US Cancer Deaths (2008 Estimates)

• Men: 291,270
• Lung & bronchus: 26%
• Colon & rectum: 15%
• Prostate: 10%
• Pancreas: 6%
• Leukemia: 6%
• Liver & intrahepatic bile duct: 4%
• Esophagus: 4%
• Non-Hodgkin lymphoma: 3%
• Urinary bladder: 3%
• Kidney: 3%
• All other sites: 23%
• Women: 273,560
• Lung & bronchus: 23%
• Breast: 15%
• Colon & rectum: 10%
• Pancreas: 6%
• Ovary: 6%
• Leukemia: 4%
• Non-Hodgkin lymphoma: 3%
• Uterine corpus: 3%
• Multiple myeloma: 2%
• Brain/ONS: 2%
• All other sites: 31%

Systematic Approach to Diagnose Hematologic Neoplasms

• CBC (Complete Blood Count), CBP (Complete Blood Picture)
  • WBC total, differential count, left shift?
  • Peripheral blood film: evaluate the cellular constituents and search for abnormalities (abnormal cells?).
• Special staining (SBB, MPO, NSE, etc).
• Bone marrow aspiration:
  • Assess the cellularity
  • M:E ratio
  • Percentage of blast cells
  • The maturation and differentiation of various cell lineage (Lymphoid, Myeloid).
• Bone marrow biopsy (trephine biopsy):
  • in solid tumors (as lymphomas with invasion to Bone marrow)
  • in cases that Bone marrow aspiration not possible (AML-M7 “due to fibrosis”, Hairy cell Leukemias, etc)
  • in cases with a compact bone marrow “very high cellular proliferation and a dry tap”
• Immunophenotyping:
  • Use of cell surface, cytoplasmic, or nuclear markers to define cell’s character and origin (phenotyping)
  • BY Flow cytometry and Immunohistochemistry studies.
• Genetics studies
  • Cytogenetics findings
  • Molecular genetics studies

CML

• Epidemiology of CML
• Cytogenetics and Molecular Biology of CML
• Clinical Presentation and Natural History of CML
• Therapeutic options for CML

Hematopoiesis

• A process by which blood-cell lineages are produced By Bone Marrow
• WBC (White blood cells or Leukocytes)
  • Myeloid lineages
  • Lymphoid lineages
  • Granulocytes- Massively expanded in CML.

Hematopoietic Differentiation

• Stem cell are capable of
  • Self- renewal
  • Differentiation
• Differentiation and proliferation is controlled by molecular signals that originate in
  • Contact with Stromal cells in the bone marrow
  • Growth Factors

Leukemia

• Group of malignant disorders of the hematopoietic tissues characteristically associated with increased numbers of white cells in the bone marrow and / or peripheral blood

Etiology of Leukemias

• Acute leukemias
  • Single cell mutation with “freezing” farther cell’s differentiation and maturation in early stages of development (e.g. stem cell)
• Chronic leukemias
  • The abnormal mutated (or transformed) cells will retain some capabilities to maturate (and differentiate) beyond the early cells (blasts) BUT they are all abnormal and useless malignant cells.

MPN (Myelo-proliferative Neoplasms)

• The type of disorder is often based on the predominant cell line that is affected, but because blood counts are often abnormal in more than one cell line, diagnoses based upon blood counts alone may be inaccurate.
• Four Main MPNs:
  • Chronic Myelogenous Leukemia (CML)
  • Polycythemia Vera (PV)
  • Essential Thrombocytosis (ET)
  • Primary Myelofibrosis (PMF)
• Additional MPNs:
  • Systemic Mastocytosis
  • Hypereosinophilic Syndrome
  • Chronic Myelomonocytic Leukemia
  • Chronic Neutrophilic Leukemia
  • Chronic Eosinophilic Leukemia

MPN Overview

• In CML, the predominant feature is a leukocytosis with a left shift. A mild anemia, normal to elevated platelet count, and a peripheral blood basophilia is often seen.
• In PV, the predominant features are elevated red blood cell indicies (RBC count, hemoglobin, and hematocrit). Patients often also have a mild leukocytosis and thrombocytosis.
• In ET, the predominant feature is an elevated platelet count. Patients also often have a mild leukocytosis and polycythemia.
• In PMF, the predominant feature is evidence of extramedullary hematopoiesis in the form of hepatomegaly, splenomegaly, and lymphadenopathy. Patients often have a mild anemia, but their WBC and platelet counts can be quite variable. Leukoerythroblastosis (tear drops, nucleated RBCs and early myeloid progenitors (including blasts) are often seen in the peripheral blood.

Epidemiology of CML

• Approximately 5,050 cases in the U.S. in 2009 (11% of all leukemias) with an incidence that increases significantly with age (median age ~ 55)
• Risk Factors include:
  • prior high dose radiation exposure (WW II / Chernobyl / etc…)
  • exposure to certain organic solvents (benzene)
  • age
  • gender (male > female)
• A very small percentage (< 0.1%) of individuals can express Bcr-Abl but not develop CML (wrong cell of origin, multiple genetic mutations leading to non-viability, immune surveillance)

CML - Pathophysiology – the Philadelphia Chromosome

• 46,XY,t(9;22)(q34;q11.2)

Chromosomal Changes in Cancer Cells

• Terminal Deletion
• Ring Chromosome
• Robertsonian Translocation
• Deletion
• Reciprocal translocation
• Insertion
• Inversion
• Isochromosomes
• Duplication

Bcr-Abl and CML

• The Philadelphia Chromosome results when a piece of chromosome #9 switches places with a piece of chromosome #22. The translocation forms an extra-long chromosome *9 (called der 9) and an extra-short chromosome #22, which is the Philadelphia chromosome that contains the abnormal, fused BCR-ABL gene.

Multiple Breakpoints in Bcr-Abl

Pathophysiologic Result of the Expression of Bcr-Abl

• Bcr-Abl expression alone is necessary and sufficient for the development of CML

Chronic Myeloid Leukemia Clinical Presentation

• Asymptomatic (~ 30%)
• Fatigue, weight loss, fever
• Abdominal fullness, pain and/or early satiety due to splenomegaly (~ 50-90%)
• Easy bruising and purpura
• Leukostasis
• Pulmonary symptoms
• Neurologic symptoms

CML – Peripheral Blood and BM Findings

• Peripheral smear can only give a presumptive diagnosis of CML [you need to confirm the t(9;22)]:
  • leukocytosis with a ‘left shift’
  • normocytic anemia
  • thrombocytosis in 50% of pts
  • absolute eosinophilia with a normal % of Eos.
  • absolute and relative increase in basophils
  • LAP score is low (not frequently employed)

Diagnostic Considerations in Chronic Myeloid Leukemia

• Karyotyping in CML
  • Allows for the diagnosis of CML
  • Requires a bone marrow aspirate for optimal metaphases
  • Allows for evaluation of clonal evolution as well as additional chromosomal abnormalities in the non-Ph+ clones
  • Occasional cryptic and complex karyotypes can result in the missed identification of the t(9;22)
• Demonstrating the presence of the t(9;22) or its gene product is absolutely essential in diagnosing a patient with CML

Diagnostic Considerations in Chronic Myeloid Leukemia

• Fluorescence in-situ hybridization (FISH) in CML:
  • Allows for the diagnosis of CML
  • Does not require a bone marrow aspirate for optimal results
  • Allows for the identification of potential duplications of the Ph chromosome
  • Allows for the identification of the loss of the der (9) chromsome
  • Allows for the identification of cryptic translocations involving Bcr-Abl

Diagnostic Considerations in Chronic Myeloid Leukemia

• Quantitative RT-PCR for Bcr-Abl in CML
  • Allows for the diagnosis of CML
  • Does not require a bone marrow aspirate for optimal results
  • Can quantify the amount of disease
  • Allows for the identification of cryptic translocations involving Bcr-Abl
  • Many primers sets only detect the p190 and/or the p210 translocation and may miss the p230 or alternative translocations

Disease Diagnosis and Monitoring in CML

• Test, Target, Tissue, Sensitivity (%), Use
  • Cytogenetics, Ph chromosome, BM, 1-10
    • Confirm diagnosis of CML
    • Evaluate karyotypic abnormalities other than Ph chromosome (ie, clonal evolution)
  • FISH, Juxtaposition of bcr and abl, PB/BM, 0.5-5
    • Confirm diagnosis of CML
    • Routine monitoring of cytogenetic response in clinically stable patients
    • Routine measurement of MRD
  • RT-PCR, bcr-abl mRNA, PB/BM, 0.0001-0.001
    • Routine measurement of MRD
    • Determine the breakpoints of the fusion genes

Therapeutic Options in Chronic Myeloid Leukemia

History of CP-CML Therapies

• Interferon – α +/- AraC
• Hydrea, or radiation therapy or Busulphan
• intensive chemotherapy
• early Interferon – α trials

Imatinib (Gleevec, Novartis)

• a small molecule tyrosine kinase inhibitor

Treatment Milestones for CML

• Definitions of Responses to Treatments
  • Hematologic Response
    • Complete Hematologic response
      • Normal PB counts (WBC < 10 and plt < 450)
      • Normal WBC differential
      • No Dz symptoms
      • Normalization of the size of the liver and spleen
  • Cytogenetic Responses: Ph+ Metaphases
    • complete: 0%
    • partial: 1% - 35%
    • minor: 36% - 65%
    • minimal: 66% - 95%
    • none: 96% - 100%
  • Molecular Responses: ratio of Bcr-Abl/Abl
    • Major Molecular Response
      • 3-log10 reduction from initial diagnosis sample (i.e. 25 →0.025)

Imatinib has Revolutionized the Treatment of CML – IRIS Trial1

• Newly diagnosed CML patients were randomized to receive either Imatinib 400 mg daily or Interferon-α at approximately 5X10^6 U/day as well as Ara-C 20 mg/m2 d1-10 q 8 days.
• Graph shows outcomes of 553 pts randomized to Imatinib.
• 96% , 98%, 85%, 69%, 92%, 87%

Treatment Options for Resistant Disease

• Dose Escalation of imatinib
• Second Generation TKIs (Tyrosine Kinase Inhibitors)
• Bone Marrow Transplant
• Clinical Trial Participation

LYMPHOMAS

WHO/REAL Classification of Lymphoid Neoplasms

• B-Cell Neoplasms
  • Precursor B-cell neoplasm
    • Precursor B-lymphoblastic leukemia/lymphoma (precursor B-acute lymphoblastic leukemia)
  • Mature (peripheral) B-neoplasms
    • B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma
    • B-cell prolymphocytic leukemia
    • Lymphoplasmacytic lymphoma
    • Splenic marginal zone B-cell lymphoma (+ villous lymphocytes)
    • Hairy cell leukemia
    • Plasma cell myeloma/plasmacytoma
    • Extranodal marginal zone B-cell lymphoma of MALT type
    • Nodal marginal zone B-cell lymphoma (+ monocytoid B cells)
    • Follicular lymphoma
    • Mantle cell lymphoma
    • Diffuse large B-cell lymphoma
    • Mediastinal large B-cell lymphoma
    • Primary effusion lymphoma
    • Burkitt’s lymphoma/Burkitt cell leukemia
• T and NK-Cell Neoplasms
  • Precursor T-cell neoplasm
    • Precursor T-lymphoblastic leukemia/lymphoma (precursor T-acute lymphoblastic leukemia
  • Mature (peripheral) T neoplasms
    • T-cell chronic lymphocytic leukemia / small lymphocytic lymphoma
    • T-cell prolymphocytic leukemia
    • T-cell granular lymphocytic leukemiaII
    • Aggressive NK leukemia
    • Adult T-cell lymphoma/leukemia (HTLV-1+)
    • Extranodal NK/T-cell lymphoma, nasal type#
    • Enteropathy-like T-cell lymphoma
    • Hepatosplenic γδ T-cell lymphoma
    • Subcutaneous panniculitis-like T-cell lymphoma
    • Mycosis fungoides/Sézary syndrome
    • Anaplastic large cell lymphoma, T/null cell, primary cutaneous type
    • Peripheral T-cell lymphoma, not otherwise characterized
    • Angioimmunoblastic T-cell lymphoma
    • Anaplastic large cell lymphoma, T/null cell, primary systemic type
• Hodgkin’s Lymphoma (Hodgkin’s Disease)
  • Nodular lymphocyte predominance Hodgkin’s lymphoma
  • Classic Hodgkin’s lymphoma
    • Nodular sclerosis Hodgkin’s lymphoma (grades 1 and 2)
    • Lymphocyte-rich classic Hodgkin’s lymphoma
    • Mixed cellularity Hodgkin’s lymphoma
    • Lymphocyte depletion Hodgkin’s lymphoma

Lymphoma

• Clonal malignant disorders that are derived from lymphoid cells: either precursor or mature T-cell or B -cell
• Majority are of B- cell origin
• Divided into 2 main types :
  • Hodgkin’s lymphoma
  • Non - Hodgkin’s lymphoma

Hodgkin’s Disease

• Histologically & clinically a distinct malignant disease
• Predominantly, B-cell disease
• Course of the disease is variable, but the prognosis has improved with modern treatment

Etiology

• ? Infection - EBV (Epstein bar virus)
• ? Environmental factors

REAL Classification

• Classic:
  • Nodular Sclerosis
  • Lymhocyte rich
  • Mixed Cellularity
  • Lymhocyte depleted
• Non-Classic
  • Nodular Lymphocyte predominant

Clinical features

• Bimodal age distribution :
  • young adults ( 20-30 yrs) & elderly (> 50yrs)
• May occur at any age
• M > F
• Lymphadenopathy:
  • most often cervical region
  • asymmetrical, discrete
  • painless, non-tender
  • elastic character on palpation ( rubbery)
  • not adherent to skin
  • fluctuate in size
• Contiguous spread via the lymphatic chain eg.involvement of abdominal & thoracic LNs
• Extra nodal disease - rare
• Hepatospleenomegaly
• Constitutional symptoms ( B symptoms )
  • Night sweats,
  • sustained fever > 38 degree celsius,
  • loss of weight >10% of body weight in 6 mo
• Fever sometimes cyclical (‘Pel-Ebstein fever’)
• Pain at the site of disease after drinking alcohol
• Pallor
• Pruritis (Itching)
• Symptoms of Bulky (>10 cm) disease

Staging

• Stage I : Involvement of single LN region (I) or extra lymphatic site (IAE )
• Stage II : Two or more LN regions involved (II) or an extra lymphatic site and lymph node regions on the same side of diaphragm
• Stage III : Involvement of lymph node regions on both sides of diaphragm, with (IIIE) or without (III) localized extra lymphatic involvement or involvement of the spleen (IIS) or both (IISE)
• Stage IV : Involvement outside LN areas (Liver, bone marrow)
• A : Absence of ‘B’ symptoms
• B : B symptoms present

Non Hodgkin’s lymphoma

• Incidence is increasing [NHL>HD]
• Median age of presentation is 65-70 yrs
• M>F
• More often clinically disseminated at diagnosis
• B-cell-70% ; T-cell-30%
• Follicular Non-hodgkin’s Lymphoma linked to cMyc and MYB activation.

Diagnosis and staging

• Similar to HD plus
• Bone marrow aspirate & trephine
• Immunophenotyping : Monoclonal antibodies directed against specific lymphocyte associated antigens B cell antigens ( CD 19, 20, 22); T cell antigens ( CD 2, 3, 5 & 7)
• Immunoglobulin determination: Ig G / IgM protein marker
• HIV

Classification

• Low grade
  • Proliferation: Low
  • Course: Indolent
  • Symptoms: -ve
  • Treatment: Not curable
• High grade
  • High
  • Rapid, fatal(un-Rx)
  • +ve
  • Potentially Curable
• Staging Similar to HD

Management

• Low grade:
  • Asymptomatic : No treatment ;
  • Radiotherapy for localised disease (Stage 1);
  • Chemotheraphy: mainstay is Chlorambucil; Initial response good , but repeated relapses, median survival 6-10 yrs;
  • Newer: Fludarabine, 2-CdA (Chlorodeoxyadenosine)
  • Monoclonal antibody: Rituximab
  • SCT/BMT

Prognosis

• Low grade : Median survival –10 yrs
• High Grade: Increasing age, advanced stage, concomitant disease, raised LDH, T- cell phenotype : Poor prognosis

AGGRESSIVE SUBTYPES

• NON-HODGKINS LYMPHOMA
• "small non-cleaved" = Burkitt's lymphoblastic Propensity for marrow, blood, and CNS involvement Rapid growth Cure possible with aggressive combination chemotherapy

BURKITT LYMPHOMA

• <1% of NHL
• Median age 31; some cases in children – Same as L3 ALL
• Most present with localized disease, about 40% with disseminated disease. Extranodal involvement common (up to 80% of pts)
• Very fast-growing, most have high LDH
• CD20+, CD10+, CD5-, tdT-
• t(8;14), t(2;8), t(8;22) with overexpression of C-MYC

Cell Cycle Clock Alterations in Human Tumors

• A plus sign indicates that this gene or gene product is altered in at least 10% of tumors analyzed. Alteration of gene product can include abnormal absence or overexpression. Alteration of gene can include mutation and promoter methylation. More than one of the indicated alterations may be found in a given tumor.
• Tumor type
  • Mammary carcinoma
  • Glioblastoma
  • Lung carcinoma
  • Pancreatic carcinoma
  • Gastrointestinal carcinoma
  • Endometrial carcinoma
  • Bladder carcinoma
  • Leukemia
  • Head and neck
  • Lymphoma
  • Melanoma
  • Hepatoma
  • Prostate carcinoma
  • Testis/ovary
  • Osteosarcoma
  • Other sarcomas

Key Concepts

• Genetic rearrangements – specifically translocations as a mechanism for cancer.
• Tissue specific phenotypes caused by these translocations
  • Why the effect is restricted to blood or lymph cells?
• The differentiation of myeloid and lymphoid cells.
• CML:
  • Presenting phenotypes, genetic cause [Philadelphia Chromosome], diagnostic criteria
  • BCR-ABL fusion gene- its origin and its role in initiation of CML
  • Treatment options : the GLEEVEC story: how does it work as an tyrosine kinase inhibitor?
• B-cell diseases:
  • Hodgkin’s Lymphoma (Genetic cause : EBV infection)
  • Non-Hodgekin’s Lymphoma (cMYC, MYB oncogene)
  • Burkitts Lymphoma (translocation leading to cMYC overexpression)
  • Treatment options for B-cell diseases.