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10/6/2016 LYMPHOMA & LYMPHOPROLIFERATIVE DX BACKGROUND Lymphomas and LPD’s are neoplasms of T, B, or NK lymphoid cells and their precursors Although having different characteristics from their KAREN FERREIRA MS,MLS (ASCP) ASSOCIATE SCIENTIFIC DIRECTORHEMATOLOGY LIFESPAN ACADEMIC MEDICAL CTR 10/19/2016 normal counterparts, the neoplastic cells of many lymphoid diseases have the features of lymphoid cells at a particular stage of differentiation Neoplastic lymphoid cells can have the characteristics of lymphocytes that normally reside in a particular organ or tissue Neoplastic lymphocytes tend to home to the tissues and specific locations where their normal counterparts reside Molecular Basis of Lymphoid Neoplasm Lymphoid Neoplasm Lymphoid neoplasms arise as a result of a series of mutations in a single lymphoid cell This is usually in a committed lymphoid cell (T, B, NK) Mutations are variable but usually involve oncogenes and the loss of tumor suppressor genes Standard cytogenetic analysis can diagnose these translocations, deletions or inversions An understanding of the normal immune system is necessary to understand the nature of LPD’s/lymphoma Normal Immune System Organs Bone marrow Lymph nodes Spleen Thymus Liver GI tract Mucosal-associated lymphoid tissue (MALT) Immune System The components of the immune system are connected by lymphatic channels and the blood stream Other cells involved Neutrophils (phagocytes) Monocytes/macrophages (APC’s) 3. Dendritic cells (APC’s) Innate immunity-does not require prior antigen exposure-immediate response-phagocytic cells, NK cells, and plasma proteins of the complement system Adaptive immunity-response to antigen exposurespecificity and memory-delayed response 1. 2. 1 10/6/2016 Lymphoid Tissue Organs of the Immune System Primary lymphoid organs 1. Bone marrow 2. Thymus Secondary lymphoid organs Lymph nodes 2. Spleen 3. Other peripheral lymphoid tissue 1. Normal Lymph Node Structue 1. Cortex Primary follicles are composed of B-cells and dendritic cells 2. Upon antigen exposure, proliferation and differentiation of B-cells cause the primary follicle to develop into a secondary follicle comprising a germinal center surrounded by a mantle zone of small Blymphocytes 3. Outside the mantle zone many follicles have a marginal zone also comprised of B-cells 4. The network of dendritic cells in the germinal center presents antigen to B-cells 1. Cortex 2. Paracortex 3. Medulla Lymph Node Structure Paracortex 1. Occupied by T-cells 2. Surrounds the primary and secondary follicles 3. Abundant dendritic cells 2 10/6/2016 Medulla Lymph Node Structure The center of the lymph node-composed of medullary cords and sinuses 2. Medulary cords T-cells B-cells Plasma cells Macrophages 1. Lymph Node Structure Lymph Node Lymph-derived from interstitial fluid and containing a variable number of lymphocytes, is brought to the lymph nodes by afferent lymphatics and is transported from the lymph nodes by an efferent lymphatic system Lymphs are also brought to the nodes by its arterial supply Lymph Node Lymphocytes recirculate from the nodes or other lymphoid tissue through the lymphatics and the blood stream back to lymphatic tissue Homing of lymphs to tissues similar to those from which they originated (skin, GI) is usual Normal B-cell Development A hematopoietic stem cell in the bone marrow gives rise to a B-cell precursor and then to a naïve B-cell, which migrates either to secondary lymphoid tissue such as a lymph node primary follicle or medulla If the B-cell is presented with antigen by a dendritic cell or macrophage further development occurs A naïve (IgM or IgD) B-cell in the primary follicle responds to antigen by class switching and migration to the mantle zone The mantle zone B-cell then migrates back into the germinal center and transforms to a centroblast and then a centrocyte within what is now a secondary follicle containing a germinal center 3 10/6/2016 B-cell development cont. Benign Reactive Lymph Node These germinal center cells undergo a somatic hypermutation before migrating to the marginal zone and then the blood stream Post-germinal center B-cells become memory cells in blood or tissues or plasma cells in tissues Reactive hyperplasia Reactive Lymph Node Normal T-cell Development Positive selection Hematopoietic stem cells in the bone marrow give If cortical thymocytes recognize a specific foreign rise to T-cell precursors, which enter the thymic medulla and then migrate from the medulla to the cortex Cortical thymocytes undergo positive selection peptide presented in a MHC I or II context by thymic epithelial cells they will survive-if not they undergo apoptosis Surviving cells develop into either CD4+ or CD8+ cells which migrate to the medulla 4 10/6/2016 Thymus T & B cell gene rearrangements T and B cell precursors have germline genes that are unusual in that they are divided into segments These must be assembled into functional genes by a process of deletion and rearrangement of gene segments to form the genes that encode the various immunoglobulin molecules (IgH, Igk, Igl) and similarly, the T-cell receptor genes (TCR’s α, β, γ, δ) Once a light chain gene has been effectively rearranged, immunoglobulin is expressed on the surface There is a greater degree of genetic rearrangement occuring in Bcells that make B cell lymphomas far more common than T-cells T cell gene rearrangements NK cells Most mature T-cells have a surface membrane NK cells are part of innate immunity complex composed of α and β chains of the T-cell receptor together with CD3 and either CD4 or CD8 which recognize specific peptides in an MHC context CD8 positive-MHC class I CD4 positive-MHC class II Do not undergo any gene rearrangements Immunophenotype LYMPHOID DIFFERENTIATION The genetic rearrangement that occurs in B and T cells is paralleled by alterations in expansion of surface membrane and cytoplasmic antigens 5 10/6/2016 Lymphoma/LPD There is a putative relationship between the normal stages of Bcell and T-lineage differentiation, and B- and T- cell lineage neoplasms Lymphoma Lymphoproliferative Disorders Peripheral Blood Bone Marrow Lymphoma/Lymphoproliferative Lymph Nodes Precursor lymphoid neoplasms Secondary Lymphoid Organs Mature B-cell neoplasms Mature T-cell neoplasms NK-neoplasms Hodgkin lymphoma Precursor Lymphoid Neoplasms Mature B-cell neoplasms B lymphoblastic leukemia/lymphoma Chronic lymphocytic leukemia/lymphoma T lymphoblastic leukemia/lymphoma Splenic marginal zone lymphoma Hairy cell leukemia Follicular lymphoma Burkitt’s Mantle cell lymphoma Diffuse large B-cell lymphoma 6 10/6/2016 DEFINITION-LEUKEMIA A malignant disease of hematopoietic tissue, characterized by replacement of normal bone marrow elements with abnormal (neoplastic) blood cells. Acute leukemia-proliferation of primitive cells (rapid course) Chronic leukemia-proliferation of mature, welldifferentiated cells (slow course) CLL/SLL CLL/SLL Definition A neoplasm composed of monomorphic small, round lymphocytes in the PB, BM, spleen and LN’s In the absence of extramedullary tissue involvement there must be > 5 x 10 9/l monoclonal lymphocytes with CLL phenotype CLL-Epidemiology Lymphocytosis must be present for >3 months Most common leukemia of adults in Western world The term SLL is used for cases with tissue 2-6 cases per 100,000 persons/year increasing with morphology and immunophenotype of CLL age to 12.8/100,000 at age 65 6.7% of all non-Hodgkin lymphoma CLL-sites of involvement Morphology-SLL PB, BM Lymph nodes Liver Spleen 7 10/6/2016 Case 1 Morphology 59 yr old male presents in ER WBC=171.9 x 109/l HGB=5.7g/dl Plt=33,000/cumm Lymphocytes=88% Absolute lymphocyte count=151.3 x 109/l Flow Cytometry 8 10/6/2016 Flow Cytometry Flow Cytometry Flow Cytometry ZAP-70 Zap-70 is a prognostic marker in CLL ●low levels of Zap-70→mutated IgVH genes=good ●high levels of Zap-70→unmutated IgVH genes=bad Correlate ZAP-70 with CD38 Expression Case 1-Diagnosis B-CHRONIC LYMPHOCYTIC LEUKEMIA -absolute lymphocytosis -smudge cells on PB smear may require albumin prep -most common “chronic” leukemia in Western world IMMUNOPHENOTYPE B-Cells (CD19+,CD20+DIM) that co-express CD5 CD23+ Must prove CLONALITY MONOCLONAL = LIGHT CHAIN RESTRICTION Dim kappa or lambda 9 10/6/2016 Hairy Cell Leukemia Indolent neoplasm of small mature B lymphoid cells with oval nuclei and abundant cytoplasm with “hairy” projections Involves PB, BM, and splenic red pulp Patients present with weakness and fatigue, left upper quadrant pain, fever, and bleeding HCL Splenomegaly and pancytopenia with only a few circulating neoplastic cells is common Only 2% of all lymphoid neoplasms HAIRY CELL LEUKEMIA Cytochemistry ♂:♀ 7:1 TARTRATE RESISTENT ACID PHOSPHATASE + Pancytopenia Blood films are incubated in AS-BI phosphoric acid Splenomegaly-diffuse red pulp infiltrate Bone marrow dry tap due to reticulin fibrosis. Hairy cells produce basic fibroblast growth factor (bFGF) which increases reticulin in BM TRAP and freshly diazotized fast garnet GBC. Duplicate slides are incubated with the substrate + L(+) tartrate TRAP Positive Hairy Cells Napthol AS-BI released by enzymatic hydrolysis couples immediately with fast garnet GBC forming insoluble maroon dye deposits at sites of activity The acid phosphatase of most normal and malignant cells is inhibited by tartrate but the acid phosphatase of hairy cells (isoenzyme 5) is resistant to tartrate 10 10/6/2016 IMMUNOPHENOTYPE Follicular Lymphoma B-cells CD19+, CD20bright Neoplasm comprised of follicle center (germinal CD103** center) B-cells which have a follicle pattern on the LN section Accounts for 20% of all lymphomas Predominantly involves lymph nodes But also invades BM, PB, and spleen CD25 CD11c BRIGHT MONOCLONAL sIg+ Follicular Lymphoma Lymph Node Architecture -FL Most patients have widespread disease at the time of dx-including peripheral and central (abdominal and thoracic) lymphadenopathy and splenomegaly The bone marrow is involved in 40-70% Only 1/3 of patients are in Stage 1 or II at dx Leukemic Phase of FL PB involvement of FL 11 10/6/2016 Immunophenotype Cytogenetics CD19+ Immunoglobulin light and heavy chains are CD20+ rearranged; variable region genes show extensive and ongoing somatic hypermutation T(14;18) Bcl-2 gene rearrangements CD10+ Bcl-2+ Bcl-6+ Monoclonal sIg light chain+ Prognosis Dependant on the extent of the disease at diagnosis BURKITT’S LEUKEMIA Endemic form is the most common type of childhood cancer in Africa where it is associated with EBV Sporadic form found in Western world 300 cases in the USA/year Male:female 2-3:1 T(8;14) T(8:14) In 90% of cases of Burkitt’s a reciprocal translocation has moved the proto-oncogene cmyc from chromosome 8 to a location close to the enhancers of HC gene on chromosome14 12 10/6/2016 Burkitt’s Uncontrolled mitosis results in a clone. Other translocations include: t(2;8) kappa t(8;22) lambda Touch prep Burkitt’s lymphoma Burkitt’s starry sky Starry night Starry sky 13 10/6/2016 Immunophenotype Case 5 B-cells (CD19+,CD20+) 70 year old male CD10 positive WBC= 17.9 x 109/l CD34 never positive HGB= 15.7g/dl TdT never positive PLt= 174,000/cumm Always with sIgM and monoclonal light chain 71% lymphs restriction Morphology- lymphs are basophilic and possess a single prominent nucleolus PB Morphology (10x) Flow Cytometry 14 10/6/2016 Flow Cytometry Flow Cytometry Molecular Biology Case 5- Diagnosis Molecular studies are positive for TCR beta and TCR gamma gene rearrangement Mature Clonal T-Cell Lymphocytosis Consistent with T-Cell Prolymphocytic Leukemia Flow cytometry of PB lymphs shows 92% T-cells expressing CD2,CD3,CD4,CD5,CD7,TCRαβ Recommend HTLV-1, cytogenetic studies and bone marrow evaluation as clinically indicated Infection and Cancer Infection contributes to 15-20% of all human cancers Infection can cause long-term inflammation, suppress a person’s immune system, or directly affect a cell’s DNA Bacterial, viral, and parasitic infections have all been linked to cancer Viral Infection and Cancer Human papilloma virus-cervical cancer Hepatitis B and C- liver cancer Human herpes virus 8 (HHV-8)- Kaposi’s sarcoma Feline leukemia virus Simian virus 40-mesothelioma in monkeys 15 10/6/2016 Viral Infection and Leukemia EBV endemic in Africa and associated with Burkitts lymphoma (infects and immortalizes B-cells) Human T-lymphotropic virus endemic in Japan and some parts of the Caribbean basin is associated with Adult T-cell Leukemia Lymphoma (infects and immortalizes CD4+ T-cells) Viral Infection and Cancer Viruses encode proteins that that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. Background HTLV-1 is an exogenous retrovirus that infects 20-30 million people worldwide Causes ATLL and HTLV-1 associated myelopathy/tropical paraparesis (CNS disease) Infects CD4+ T-cells First human retrovirus discovered (1977) Viral Properties Deltavirus genera of the Orthoretrovirinae subfamily Spherical, enveloped retrovirus with a diameter of 80-120nm The envelope surrounds a capsid that contains two identical copies of positive-strand genome inside an electron dense core. Tax- a trancriptional activator Tax Role in viral replication Promotes accelerated cell cycle Transformation of infected cell Proliferation Activates and/or deactivates transcription from viral Obliteration of programmed cell death promoter Modulates transcription Affecting cell growth and differentiation, cell cycle control and DNA repair Known to deregulate expression of more than 100 genes Multiple Tax functions cooperate together for malignant transformation of HTLV-1 infected cells Induces expression of IL-1, which stimulates osteoclast activity 16 10/6/2016 Tax Epidemiology 10-20 million HTLV-1 carriers worldwide Endemic in Japan (15-30%) of the population. (1.2 million people infected) Caribbean basin (3-6%) South America (2%) US blood donors 0.025% HTLV-1 seropositivity. Seropositivity among US prostitutes and IV drug abusers→7-49% Transmission – 3 routes Mother to infant – mostly breast milk but also intrauterine infection 2. Sexual transmission- the rate of transmission between husband and wife over a 10 year period is 60.8% from husband to wife and 0.4% from wife to husband 3. Parenteral transmission – blood transfusion or IV drug use 1. Adult T-Cell Leukemia Lymphoma Generalized LN, PB, and skin involvement by pleomorphic tumor cells, ranging from small to large in size with characteristic “flower” shape nuclei Patients present with lytic bone lesions and hypercalcemia Rapidly progressive course- short survival ATLL- 4 clinical presentations Studies indicate a long latency period for HTLV-1 and lifetime ATLL risk of less than 5% in infected individuals Acute Lymphomatous Chronic Smouldering ATLL- Acute 55-60% of cases Severe PB lymphocytosis with numerous “flower” cells Hypercalcemia Hepatoslenomegaly Elevated serum LDH Median survival is 6 months despite multiagent chemotherapy 17 10/6/2016 ATLL – “Flower” Cells ATLL - Chronic 15-20% of cases Normal to slightly elevated lymphocyte count with some circulating “flower” cells Median survival is 2 years, but most patients die within 5 years due to progression to acute or lymphomatous phases Diagnosis Documentation of HTLV-1 infection by serologic analysis or detection of clonal integration of HTLV-1 genome into neoplastic cells by molecular techniques Vigilant morphologic analysis of PB lymphocyte morphology Documentation of specific ATLL immunophenotype (flow cytometry or immunohistochemistry) Case Study 6 ATLL- Immunophenotype CD2+, CD3+ (dim), CD4+, *CD25+ (IL-2 receptor), aberrant loss of pan T-cell marker CD7 *When a T-cell is activated, it secretes IL-2, at the same time it upregulates the IL-2 receptor on its’ surface. The secreted IL-2 the binds to the upregulated IL-2 receptor and results in cell division PB Wright’s stain x1000 60 yo male with weakness referred to Heme/Onc by PCP Calcium 15.5 mg/dL –hypercalcemia Small palpable left cervical LN Liver enzymes increased WBC 90.0 x 109/L with 6% blasts, 8% atypical lymphocytes, 63% lymphocytes 18 10/6/2016 PB PB PB PB PB PB 19 10/6/2016 BM asp Wright’s stain x1000 BM bx H&E stain x200 BM bx Immunophenotyping Flow Cytometry Flow Cytometry 20 10/6/2016 Case 7 Flow Cytometry CD25 positivity is hallmark for ATLL 57 year old female Post kidney transplant WBC= 6.9 x 109/l HGB=7.5g/dl Plt=386,000/cumm Normal diff Pleural Effusion- nucleated cells=1340/cumm Pleural Fluid (20x) 21 10/6/2016 Viral load The pleural fluid has 2 million copies/ml of EBV Molecular Biology Heavy chain gene rearrangement is detected by PCR compared with 277,000 in the blood Case 7- Diagnosis EBV positive post transplant lymphoproliferative malignancy with morphologic and immunophenotypic evidence of plasma cell differentiation Basophilic cells, large and small. CD45dim, CD19,CD20,CD22,CD52,CD30 are negative. T-cell markers are negative. CD38, CD138, and CD56 are strongly +. sIG light chains are neg, but there is cIg kappa restriction. CBC Interpretation RBC 3.45x10^12/L HGB 11.4g/dL HCT 34.1% 0.6x10^9/L MCV 99.0fL 2.2x10^9/L MCH 33.1pg 0.1x10^9/L MCHC 33.5g/dL RDW 16.9% Platelets 62x10^9/L WBC 3.0x10^9/L Absoulute Values: Neutrophil Lymphocytes Monocytes Eosinophils 0x10^9/L Basophils 0x10^9/L Case #3 Patient History A 60 year old female presents to her physician with noticeably easy bruising and profound fatigue. History of Plavix use in the past. Not currently taking any medication besides baby aspirin. Past Medical History: Remarkable for arthritis, hypertension and hypothyroidism. A year ago, she had a normal white count, normal hemoglobin and platelet count 99,000, slightly below normal value. Peripheral Blood Review Pancytopenia with an absolute monocytopenia as well as neutropenia. Absolute lymphocyte number is within normal range however, a large subset show medium sized nucleoli and abundant cytoplasm with numerous hair-like projections. Rare nucleated RBC seen on scanning. 22 10/6/2016 Peripheral Blood Morphology Peripheral Blood Morphology Peripheral Blood Morphology Peripheral Blood Morphology Bone Marrow Aspirate Smear Bone Marrow Sinusoidal Morphology No cellular bone marrow spicules for evaluation- poor aspirate sample or dry tap? Predominant cell type in sinusoidal blood: Lymphocytes; most being small to medium sized with slightly larger nuclei and occasional nucleoli. Significantly decreased myeloid lineage. Erythrocyte and megakaryocyte series are essentially absent in the sinusoidal blood. 23 10/6/2016 Bone Marrow Sinusoidal Morphology Bone Marrow Sinusoidal Morphology Cytochemical Stains Tartrate Resistant Acid Phosphatase stain Tartrate Resistant Acid Phosphatase stain (TRAP): Acid Phosphatase (isoenzyme 5) is resistant to tartrate. A specific subset of lymphocytes as well as histiocytes contain this acid phosphatase compound and are therefore resistant to inhibition, and will demonstrate positivity. Gaucher Disease is another clinical condition in which TRAP staining is positive. Our TRAP stain revealed that the subset of lymphocytes with the hair-like projections are positive for TRAP staining. Tartrate Resistant Acid Phosphatase stain Bone Marrow Biopsy The decalcified bone marrow biopsy consists of a core of trabecular bone that measured 0.8cm in length. The cellularity ranges from 50-100%, overall approximatley 70% cellular. Architecture of the bone marrow is predominantly effaced by lymphocytes with abundant cytoplasm giving a “fried egg” appearance. The lymphocytes replace >75% of the marrow, leaving behind only pockets of hematopoietic marrow. 24 10/6/2016 Immunohistochemical Stains Core biopsy CD20 PAX-5 Annexin A1 All the above stains highlight a the specific lymphocyte population in question with near effacement of the marrow. CD20 CD3 FLOW CYTOMETRY HISTOGRAMS-Gating Monoclonal Antibodies-T and B-cells 25 10/6/2016 ISOLATING B-CELLS ISOLATING B-CELLS B-CELLS SURFACE IG LIGHT CHAINS Over View Slow-growing lymphoid malignancy. Future procedures include a CT of the chest, abdomen and pelvis to assess for adenopathy, Treatment plan: 1-week treatment with Cladribine as a 2-hour infusion and then 4 weeks later give 8 consecutive treatments with Rituximab. 26