Lymphocytes and Lymphoma

Question 1

List and describe the Lymphoid system components

Answer 1

T cells
o Major component of cell mediated immunity
o Develop in thymus and traffic to lymph tissues
Helper T cells (CD4+)
• Recognizes Ag presented in context of MHC class II molecules
• Coordinates immune response by involving other cells
Cytotoxic T cells (CD8+)
• Kills cells presenting Ag in context of MHC class I molecules

NK Cells
o Kill cells with low MHC class I expression
o Have Fc Receptors
o Participate in antibody dependent cellular cytotoxicity

B cells
o Major cells for developing humoral immunity
o Develop in marrow and traffic to nodes/lymph tissues
• Pre-programmed when leave marrow to recognize Ag
o After recognizing Ag they self select best clones to become memory cells and plasma cells
o Plasma cells are immunoglobulin secreting cells
• IgM, IgG, IgA, IgD, & IgE

Question 2

Define lymphopenia

Answer 2

lymphocyte count <1,000/μl
o Occurs in aplastic anemia, AIDS, certain immunodeficiency diseases, after treatment with corticosteroids and immunosuppressants, in associated with some lymphomas (advanced Hodgkin Lymphoma)

Question 3

Define lymphocytosis

Answer 3

lymphocyte count >5,000/μl
o Children = more often reactive = occurs with acute infections: EBV-associated infectious mononucleosis and Bordetella pertussis infection
o Older adults = chronic lymphocytic leukemia

Question 4

Define lymphoma

Answer 4

Heterogeneous group of lymphoproliferative malignancies
• Results from clonal expansion of tumor cells derived from B, T, or NK cells
• 60+ different histologic subtypes
• 85%-90% in the US are derived from B cells

Range of clinical presentations:
• Asymptomatic pick up on routine blood work
• Painless adenopathy
• Emergent medical problem

Variable natural histories, therapeutic responses, prognoses

Question 5

Normal Lymphoid protein antigens

Answer 5

B cells: CD19, CD20, surface Ig

Plasma Cells: CD138, Cytoplasmic Ig

T cells: CD2, CD3, CD5, CD7; CD4 for helper cells and CD8 for cytotoxic T cells

NK cells: CD2, CD16, CD56; CD158 (killer inhibitory receptors/ KIRs

Question 6

B-cell Biology and Lymphoma relationship:

Answer 6

Normal B cell response to Ag create intense genomic pressure:
•	More receptor editing, somatic hypermutation, and class switching → more chance for error/mutation

Most lymphomas arise from B cells which have trafficked through the germinal center

Greater Ag stimulus increases risk for the development of lymphoma:
• Chronic infections (EBV, H. pylori, Hepatitis C)
• Sjogrens syndrome, Hashimotos thyroiditis
• Environmental exposures (chemicals, pesticides, etc.)

Question 7

Normal Lymph node morphology

Answer 7

Cortex contains primary follicles
-Contain naive B cells

Reactive = Germinal Centers:

• Surrounding mantle zone (resting B cells)
• Inner zone: proliferating B cells and somatic hypermutation –> selection –> generation of memory cells, plasma cell precursors and class switching

T cell paracortical zone

Medulla

Question 8

Causes of Lymphocytosis

Answer 8

Infection:
-EBV, CMV, Hantavirus, early HIV, brucellosis, toxoplasmosis, TB, pertussis, other

Drug:
-Dilantin

Other:
-Smoking (polyclonal B cell lymphocytosis); post-vaccination

Endocrine:
-Thyrotoxicosis, acute stress (transient)

Malignant:
-CLL, large granular lymphocytic leukemia, peripheralization of other lymphoid malignancies like mantle cell, marginal zone, and follicular lymphoma

Question 9

EBV (infectious mononucleosis): symptoms & hematologic findings

Answer 9

Symptoms:
• Fever, sore throat, profound fatigue, enlarged lymph nodes in neck and axillae, rash, splenomegaly, hepatomegaly (less often)
• Usually last 1-4 weeks

More complicated symptoms
• Splenic rupture, aseptic meningitis, respiratory distress from enlarged tonsils, severe hepatitis
• Rare

Hematologic findings:
• Lymphocytoiss with predominance of atypical lymphocytes = Mainly CD8+ T cells that react to EBV-produced antigens
• Anemia = rare
• Mild reduction in platelets (25% cases)
• Liver enzyme elevation (80% cases)

Question 10

EBV (infectious mononucleosis): diagnosis

Answer 10

Monospot test
• Rapid but not very specific
• Tests for presence of heterophile Abs (released by activated B cells → agglutination of horse RBC or hemolysis of ox RBC)

Assay for anti-EBV antibodies
• More specific test
• Early in infection = rise in IgM against viral capsid antigen and Abs against “early antigen”
• Later in infection = IgG against viral capsid appears = persists for life
• After several weeks = Ab against Epstein-Barr nuclear antigen develops

Quantitative PCR assays
• EB virions in blood

Question 11

EBV (infectious mononucleosis): phases & immune response

Answer 11

o	Phases: 
1) Lytic (epithelial cells, B cells)
•	Active viral replication and lysis
•	Most genes active
2) Latent (B cells)
•	DNA circularizes forming episome
•	10 genes expressed
•	Viral replication a rare phenomenon
•	B cells are transformed (immortalized)

Immune Response
Humoral response mostly useful for dx
• Monospot test detects heterophile Abs
Vigorous cellular response keeps transformed immunoblasts under control
• CD8+ cytotoxic T cells proliferate & expand (atypical lymphs)
• CTL’s efficiently clear infected immunoblasts
Response is virtually watertight in preventing proliferation of virally transformed immunoblasts

Immune Surveillance
• Virus persists in the small resting B cell fraction
• Cells with only the EBNA 1 program
• Cells remain invisible to the immune system
• No tendency to expand, proliferate, or accumulate
In normal adults, about 1 x 106 B cells are infected
• Tends to remains stable over time
• Unless immune system disrupted

Question 12

EBV (infectious mononucleosis): role in post-transplant lymphoproliferative disorders (PTLD)

Answer 12

• EBV driven proliferation of infected B cells
• Occurs in setting of iatrogenic T cells suppression
• Spectrum of clinically and morphologically heterogeneous lymphoid proliferations
• Proliferations progress from polyclonal to oligoclonal to monoclonal

PTLD Risk Factors
• Degree of immunosuppression
• Frequency of rejection episodes
• Risk highest first year after transplant
• Later episodes less likely to be EBV related

Important treatment is reduction in immunosuppression

Question 13

Hodgkin’s Lymphoma: epidemiology

Answer 13

o Bimodal distribution

o Often seen in young adults

Question 14

Hodgkin’s Lymphoma: pathology

Answer 14

o B cell lymphoma

Characteristic Reed-Sternberg cells
• Large cells with bilobed nuclei
• Large eosiniphilic nucleolus = “owl-eyed” appearance
o See RS cells in background of benign cells of mixed lineage
o May have fibrotic bands and sclerosis of lymph node capsule (Nodular Sclerosing HL)

A “crippled” germinal center B cell = can’t make functional Ig
• Somatic mutations result in stop codon (no sIg)
• No apoptotic death → malignant transformation

Does NOT have normal B cell surface antigens:
• NO CD20, CD19, CD45 (unlike most B cells)
• Express CD30

Potent cytokine producing cells → inflammatory reaction of neutrophils, eosinophils, plasma cells

o Unclear how this occurs (EBV present in 50% of cases)
o Unclear how cells end up with RS phenotype

Question 15

Hodgkin’s Lymphoma: clinical features & lab findings

Answer 15

Often seen in young adults
• Present with painless lymphadenopathy (often in neck or supraclavicular fossa)

Wide variety of presentations
•	B symptoms (fevers, night sweats, weight loss)
•	Pruritis
•	Cough/SOB
•	Pain
•	Painless adenopathy

Lab findings:
•	Normocytic anemia
•	Lymphopenia
•	Elevated ESR
•	Elevated LDH

Question 16

Hodgkin’s Lymphoma: staging

Answer 16

Typical staging results:
o Stage I: node involvement in one lymph area
o Stage II: disease involving 2 or more nodal areas confined to one side of diaphragm
o Stage III: nodal disease above and below diaphragm
o Stage IV: widespread involvement including disease outside lymph node system

o Most often disease is localized to above the diaphragm
o Common to have extensive mediastinal disease
o Tends to spread to contiguous nodal groups (Unlike NHL)

Question 17

Hodgkin’s Lymphoma: treatment

Answer 17

o Dictated mainly by where the disease is located rather (results of staging) than the exact histologic subtype
o 4 subtypes of classical Hodgkins

For stage I and II:
• Abbreviated chemotherapy + radiotherapy
More advanced disease:
• Repeated cycles of combination chemotherapy

Question 18

Hodgkin’s Lymphoma: prognosis

Answer 18

IPS score:
o	Based on number of risk factors:
Age greater than 45
Male
Low albumin
Anemia
Stage IV
WBC over 15k
Lymphopenia 

Highly curable with current treatments (5 year survival rates):
• Stage I and II: 85-95%
• Stage III: 70-85%
• Stage IV: 55-70%

Question 19

Non-Hodgkin Lymphoma (NHL): epidemiology

Answer 19

o Median age of diagnosis: 60 years

Question 20

Non-Hodgkin Lymphoma (NHL): morphology

Answer 20

60ish different subtypes
• B cell (85%), T cell, and NK cell
• Histologic subtype determines patient approach

Question 21

Non-Hodgkin Lymphoma (NHL): types

Answer 21

Often widespread disease at diagnosis 
Wide variation in outcome:
•	Some cases rapidly fatal
•	Some cases readily curable
•	Some cases incurable BUT patient can live for many years with good quality of life

Types:
Aggressive NHL
• Short natural history (patients die within months if untreated)
• Disease of rapid cellular proliferation
• Potentially curable with chemotherapy

Indolent NHL
• Long natural history (patients can live for many years untreated)
• Disease of slow cellular accumulation
• Generally incurable with chemotherapy

Question 22

Non-Hodgkin Lymphoma (NHL): clinical presentation & lab findings

Answer 22

Aggressive NHL:
Symptomatic presentation:
•	Palpable lymphadenopathy 
•	B symptoms
•	Pain
•	Organ failure
•	Variety of other symptoms

Indolent NHL:
• Painless adenopathy

Overall: more prone to bacterial than fungal or viral infections

Lab findings:
• Coombs positive hemolytic anemia
• Ig deficiency
• Monoclonal gammopathy

Question 23

Non-Hodgkin Lymphoma (NHL): staging

Answer 23

o History and physical

o Lab work:
• Beta 2 microglobulin = prognostic in indolent lymphomas
• LDH = prognostic in aggressive lymphomas

o CT scans
o Bone marrow biopsy
o PET scan
o CNS evaluation (lumbar puncture and CSF exam) with aggressive lymphomas

Question 24

Non-Hodgkin Lymphoma (NHL): treatment

Answer 24

Dictated mainly by histology
• Reliable hematopathology crucial

Aggressive NHL
Combinations of drugs:
•	Anti-CD20 agents (Rituximab)
•	Cyclophosphamide
•	Vincristine 
•	Adriamycin 
•	Prednisone 
Radiotherapy to palliate localized symptomatic disease 
Cure is often the goal

Indolent NHL
• Immediate treatment does not prolong overall survival for asymptomatic low tumor burden patients
• So = watch and wait approach
When to treat?
• Constitutional symptoms
• Compromise of a vital organ by compression or infiltration, particularly the bone marrow
• Bulky adenopathy
• Rapid progression
• Evidence of transformation
Patients with symptoms or high tumor burden warrant immediate treatment
Control is the goal

Question 25

Non-Hodgkin Lymphoma (NHL): prognosis

Answer 25

Measured based on IPI (international prognostic index) stratification based on risk factors

Risk factors:
•	Age over 60
•	Pathologic classification 
•	Immunologic function 
•	Stage III or IV and mass of disease
•	Growth rate
•	Site (bad prognosis = brain lymphoma)
•	High LDH level 
•	Symptoms
•	Performance status over 1

Question 26

Describe the role of chromosome translocations in the pathophysiology of follicular NHL

Answer 26

t(14;18)
Follicular lymphoma
o Places bcl-2 oncogene from chromosome 18 → chromosome 14 under the IgH promoter
o Result: over-expression of bcl-2 → increases apoptotic threshold = cells don’t die
• Indolent lymphoma with slow accumulation of malignant cells

Question 27

Describe the role of chromosome translocations in the pathophysiology of Burkitt’s Lymphoma

Answer 27

t(8;14)
Burkitt Lymphoma
o Places c-myc oncogene from chromosome 8 → chromosome 14 under the IgH promoter
o Result: over-expression of c-myc → proliferation signal
• Aggressive malignancy with high growth rate

Question 28

Describe the role of chromosome translocations in the pathophysiology of Mantle Cell Lymphoma

Answer 28

t(11;14)
Oncogene: Bcl-1
Protein: Cyclin D1
Results in increased proliferation

Question 29

Describe the role of chromosome translocations in the pathophysiology of Large Cell Lymphoma

Answer 29

t(3;14)
Oncogene: Bcl-6
Protein: Zinc-finger TF
Results in deregulated transcription

• Aggressive
Use expression array to figure out sub-type
o Look same on histology
o BUT different outcomes:
o Germinal center B-cell like type has better survival than Activated B-cell like type

Question 30

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL): epidemiology

Answer 30

o Most common adult leukemia in Western world
o Median age 68
o Affects males more than females

Question 31

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL): clinical presentation

Answer 31

Often diagnosed on routine blood work
• Combination of immunodeficiency and propensity toward autoantibody mediated immune phenomena
• Ex: autoimmune hemolysis anemia; immune thrombocytopenia

o Isolated lymphocytosis
o Patients usually asymptomatic
o An indolent lymphoproliferative disease

Manifestations can include:
Marrow failure (anemia, thrombocytopenia, neutropenia)
Lymphadenopathy and splenomegaly
Recurrent infections due to impaired humoral immunity
• Decreased levels of normal immunoglobulins
T cell deficiency:
o Malignant B cells produce immunosuppressive cytokines (TFG-β)
o Also soluble cell surface receptors (CD27) = inhibit T cell function
• Increased incidence of 2nd malignancies = Carcinoma of bowel, lung, skin, prostate, and bladder
• Autoimmune cytopenias due to dysregulation of immune system

Minority of cases = transformation to large cell lymphoma
• “Richter’s Transformation”

Question 32

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL): pathology

Answer 32

o Large numbers of normal-appearing small lymphocytes
o Smudge cells prominent

Characteristic immunophenotype:
Monoclonal B cells with IgM + IgD (all are either kappa or lambda light chains) and CD5 (normally a T cell antigen), CD19, low amounts of CD20 and CD23

Use flow cytometry:
• Look for kappa: lambda ratio (normally 3:1)
• Look for co-expression of CD19 with CD5 (in CLL and mantle cell lymphoma)
• Expression of CD23 (positive in CLL; negative in mantle cell lymphoma)

Question 33

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL): treatment

Answer 33

If asymptomatic = not require treatment

Symptomatic
• Chemotherapy with or without Rituximab (anti-CD20 mAb)
o Incurable (except some cases after allogenic bone marrow transplant)