Clinicopathological Conference Case 39-2008 Jeremy S. Abramson, M.D. Hematology-Oncology Manjil Chatterji, M.D. Radiology Aliyah Rahemtullah, M.D.

Pathology

A 51-year-old woman with splenomegaly and anemia Presentation of Case

Jeffrey Barnes, M.D.,Ph.D. Hematology-Oncology

Presentation
51-year-old woman admitted to MGH because of anemia and splenomegaly. 2 months before admission pain in her feet, night sweats and fatigue developed .
Left upper quadrant fullness, early satiety, edema of the legs, and dyspnea on exertion; Furosemide was prescribed, but symptoms persisted.

First admission to another hospital

On examination, the spleen extended to the pelvic brim and the legs were edematous.

Labs at the Other Hospital

WBC 4.7 with nl diff
Hgb/HCT 6.1/21.1 MCV 77

IgG 284
IgA 72 IgM 329

PLTs 209K
Iron 18 TIBC 222 Ferritin 167

SPEP Faint IgM kappa band

PT 14.5 INR 1.5 PTT 64.6

Other Hospital, First Admission

Direct Coombs (DAT-IgG and DAT-Complement): negative. Two units of red blood cells were transfused CT of the chest, abdomen and pelvis:
splenomegaly, ascites, lymphadenopathy (neck, splenic hilum, paraaortic)

PTT prolongation
Did not correct with mixing with normal plasma in a 1:1 ratio Remained prolonged after 2 hours.

Three units of fresh frozen plasma and two additional units of red cells were given. Bone marrow biopsy performed and she was discharged on day 5

Bone Marrow Biopsy

Lymphoid aggregates comprising approximately 10% of the marrow cellularity, and increased interstitial lymphoid cells. Flow cytometry: CD19+ kappa+ B cells, lacking CD5, CD23, CD10, and CD103.

Hematology Visit
3 weeks before admission she saw a local hematologist
HCT stable at 29. LDH elevated at 530. Anticardiolipin IgM elevated at >100 units/ml

Referral to MGH Cancer Center

Readmission
One day before MGH admission, she was readmitted to the other hospital Increasing malaise, lower extremity edema, fever to 101.5F, and night sweats. On examination, the temperature was 98.8F; the splenomegaly was unchanged. There was 2+ edema of the legs.

Readmission labs
WBC 4.5 with 8% bands
Hgb/HCT 5.7/20.3 PLTs 174K

PT 15.5 INR 1.6 PTT 63

LDH 1030

Readmission to Other Hospital

2 units of packed, leukocyte-reduced, red cells were transfused. CT of the abdomen: splenomegaly and lymphadenopathy unchanged She was transferred to MGH.

Other History
Medical
Follicular carcinoma of the thyroid (remote):thyroidectomy and radioactive iodine. Migraine headaches, tremors, depression, anxiety.

Social
Homemaker, lived with husband and stepchild. Past tobacco, ETOH; no illicit drugs.

Family
Mother: systemic lupus erythematosus, coronary artery disease, stroke; died aged 55 years Father alive, 80s: tremors, stroke, myocardial infarction, cerebral aneurysm 3 siblings healthy.

Medications listed in handout; NKDA

Physical Examination
T101.0F, BP110/68, HR 105, RR 18, 96 % RA Mild scleral icterus Abdomen soft, no tenderness or distension.
Spleen firm and nontender, extended below the pelvic brim.

No peripheral lymphadenopathy Remainder of exam normal

Initial Hospital Course

Serum protein electrophoresis:
Small monoclonal IgM kappa band Marked decrease in normal immunoglobulin.

CT of the chest, abdomen and pelvis confirmed findings from outside hospital Two units of filtered red cells transfused. Folic acid (5 mg daily) begun. Vitamin K (total 25 mg) given subcutaneously.

Hospital Course cont.

3 additional units of red cells and 4 units of fresh frozen plasma were transfused; no correction of the PTT occurred Coagulation testing:
Mixing study: PTT did not correct with addition of normal plasma. Coagulation factors II, V, VII, X levels normal Factors VIII, IX, XI and XII decreased

Hepatitis A IgM antibody reactive; total antibody to Hepatitis A non-reactive. Peripheral blood flow cytometry: CD19+, CD20+ kappa+ B-cell population dimly co-expressing CD23 and negative for CD5 and CD10. A diagnostic procedure was performed

Differential Diagnosis Jeremy S. Abramson, M.D. Hematology-Oncology

Salient features to Differential Diagnosis

Splenomegaly Anemia Paraproteinemia Coagulopathy

Radiology Studies
Manjil Chatterji, M.D. Radiology

30 cm

B
10 cm

30 cm

Coronal (Panel A) and axial (Panel B) images show splenomegaly (30 cm in greatest length, Panel A) and scattered, slightly enlarged lymph nodes in the paraaortic and splenic hilar regions (arrows, Panel B).

Splenomegaly
Hematologic
Membrane defects Hemoglobinopathies Autoimmune cytopenias Rheumatoid arthritis Lupus Sarcoidosis

Congestion
Cirrhosis Venous thromboses Congestive heart failure Hematologic neoplasms Myeloproliferative diseases Amyloidosis Glycogen storage diseases

Rheumatologic Diseases Infiltrative diseases

Infections

Massive Splenomegaly
Thallasemia Major Infections
Visceral leishmaniasis Hyperreactive malarial splenomegaly syndrome Mycobacterium avium complex

Infiltrative diseases
Lymphoproliferative diseases Myeloproliferative diseases Gauchers disease

Massive Splenomegaly
Thallasemia Major Infections
Visceral leishmaniasis Hyperreactive malarial splenomegaly syndrome Mycobacterium avium complex

Infiltrative diseases
Lymphoproliferative diseases Myeloproliferative diseases Gauchers disease

Gauchers Disease
Mutation of Glucocerebrosidase gene More common in Ashkenazi Jewish population Autosomal recessive Variability in clinical presentation and severity Half of cases diagnosed before 10 years, but 20% over 30. Some cases diagnosed late into adulthood Presents with symptoms related to splenomegaly, hepatomegaly, anemia, thrombocytopenia, and osteopenia Monoclonal gammopathy may be present Diagnosis: Biopsy of organ or marrow

Charrow J, et al. Arch Int Med 2000. Beutler E. in Williams Hematology.

Myeloproliferative diseases
Clonal stem cell disorders resulting in proliferation of one or more cell line
Chronic Myelogenous Leukemia Polycythemia Vera Essential thrombocytosis Chronic Idiopathic myelofibrosis

Antecedent increase in one or more cell lines Late pancytopenia Peripheral evidence of extramedullary hematopoiesis and myelophthisis

Lymphoproliferative diseases with splenomegaly

Primary vs. Secondary Indolent vs. Aggressive 108 splenectomies for lymphoproliferative disease
B-cell diseases 96%
DLBCL 33.3% CLL/SLL 19.4% Follicular lymphoma 13% Lymphoplasmacytic lymphoma 9.3% Splenic marginal zone lymphoma 8.3% Mantle Cell lymphoma 6.5% Hairy Cell leukemia 6.5%

T-cell diseases 4%

Arber, et al. Modern Path 1997.

Large Granular Lymphocyte Leukemia (LGL)

Clonal disease of cytotoxic T-cells or NK-cells Presents with prominent cytopenias (neutropenia most common) +/- splenomegaly Indolent natural history Median age 60 Associated with autoimmune diseases (esp. RA) Monoclonal gammopathy may be present Diagnosis: Examination and flow cytometry of the peripheral blood and bone marrow

Hepatosplenic T-cell lymphoma

Aggressive clonal proliferation of T-cells in liver, spleen and bone marrow Prominent hepatosplenomegaly, B symptoms and cytopenias Presents in young men, median age 34 Risk factor is immunosuppression, transplant Diagnosis: Examination of bone marrow, liver, spleen

Systemic B-cell Lymphomas

Diffuse Large B-cell lymphoma Follicular Lymphoma Mantle Cell Lymphoma Small lymphocytic lymphoma/Chronic lymphocytic leukemia Lymphoplasmacytic lymphoma Marginal zone lymphoma Hairy cell leukemia

CLL/SLL
Indolent mature B-cell leukemia/lymphoma Median age 65 Male:Female 2:1 May present with lymphocytosis, adenopathy, splenomegaly, cytopenias Monoclonal gammopathy may be present Diagnosis: Examination and flow cytometry of blood, marrow or nodal disease

Lymphoplasmacytic Lymphoma/ Waldenstrms Macroblobulinemia

Lymphoplasmacytic infiltration of bone marrow, lymph nodes, spleen and liver IgM paraprotein production Symptoms due to tumor itself and IgM Anemia is common Median age 63 Very slight male predominance Diagnosis: Marrow examination and flow cytometry and IgM

Hairy Cell Leukemia

Indolent B-cell neoplasm infiltrating bone marrow, peripheral blood, and spleen Presents with pancytopenia and marked splenomegaly Monocytopenia common Median age 55 Male: Female 4:1 Diagnosis: examination and flow cytometry of peripheral blood and bone marrow

Splenic Marginal Zone Lymphoma +/- Villous Lymphocytes

Mature B-cell lymphoma with involvement of the spleen, splenic hilar lymph nodes, bone marrow and peripheral blood Distinct from other marginal zone lymphomas (extranodal MALT and nodal) May be HCV associated Small paraprotein is common, IgM > IgG Has been associated with autoimmune phenomena

Splenic Marginal Zone Lymphoma +/- Villous Lymphocytes

Median age is 68, no gender predominance Presents with symptoms of splenomegaly and anemia Anemia may be due to splenic sequestration, autoimmune destruction, or marrow infiltration Other cytopenias generally mild Minimal systemic lymphadenopathy Diagnosis: Examination and flow cytometry of blood, bone marrow, spleen in context of clinical presentation

Clinical Diagnosis
Massive splenomegaly with minimal systemic adenopathy Mature clonal B-cell population in blood and marrow CD20+CD10-CD5-CD23 Anemia and IgM paraproteinemia
SPLENIC MARGINAL ZONE LYMPHOMA

Etiology of the Anemia

General Approach to Cytopenias

Cell Production

Cell Destruction

Increased Cell Destruction or Loss

Bleeding Dilution Autoimmune destruction Mechanical destruction Splenic sequestration

Retic

Hemolytic Anemia
Increased reticulocyte count Increased lactate dehydrogenase (LDH) Undetectable haptoglobin Increased total and indirect bilirubin

Hemolytic Anemia
Intrinsic red cell defects
Membrane Hemoglobinopathy Enzyme deficiency PNH

Extrinsic non-immune
Splenic sequestration Mechanical destruction
Microangiopathic Macroangiopathic March hemoglobinuria Foreign body Oxidant drugs Toxins Osmotic lysis Infection
Malaria/Babesia Clostridial sepsis

Extrinsic Immune-mediated
Autoimmune
Warm Cold Seronegative

Alloimmune

Hemolytic Anemia
Intrinsic red cell defects
Membrane Hemoglobinopathy Enzyme deficiency PNH

Extrinsic non-immune
Splenic sequestration Mechanical destruction
Microangiopathic Macroangiopathic March hemoglobinuria Foreign body Oxidant drugs Toxins Osmotic lysis Infection
Malaria/Babesia Clostridial sepsis

Extrinsic Immune-mediated
Autoimmune
Warm Cold Seronegative

Alloimmune

Hemolytic Anemia
Intrinsic red cell defects
Membrane Hemoglobinopathy Enzyme deficiency PNH

Extrinsic non-immune
Splenic sequestration Mechanical destruction
Microangiopathic Macroangiopathic March hemoglobinuria Foreign body Oxidant drugs Toxins Osmotic lysis Infection
Malaria/Babesia Clostridial sepsis

Extrinsic Immune-mediated
Autoimmune
Warm Cold Seronegative

Alloimmune

Seronegative AIHA
3-7% of autoimmune hemolytic anemia Etiology
Low level of IgG bound to red cells below limits of detection by DAT Low affinity IgG autoantibodies IgA or IgM autoantibodies

Garrity G. Sem Heme 2005.

Autoimmune phenomena in this patient

Mildly increased IgM with monoclonal band False positive anti-Hepatitis A IgM High titer anticardiolipin IgM High titer lupus anticoagulant ? Coombs negative AIHA

Etiology of the Coagulopathy

Coagulopathy (increased PT and aPTT): Differential Diagnosis

Vitamin K deficiency due to anorexia Disseminated intravascular coagulation Hypo- or dysfibrinogenemia Acquired coagulopathy in lymphoproliferative disease
Acquired factor deficiency due to adsorption Acquired factor inhibitor Acquired Von Willebrand disease Lupus anticoagulant

Coagulopathy (increased PT and aPTT): Workup

PT corrected with Vitamin K, aPTT still prolonged Mixing study showed no correction with factor replacement Increased D-dimer Thrombin time normal. No factor inhibitor identified High titer lupus anticoagulant present

Clinical Diagnosis
Splenic Marginal zone lymphoma with an IgM paraprotein and lupus anticoagulant Hemolytic anemia due to splenic sequestration +/- Coombs negative AIHA

Diagnostic/therapeutic procedure: Splenectomy

Pathology
Aliyah Rahemtullah, M.D. Hematopathology

The bone marrow biopsy specimen contains multiple ill-circumscribed and nonparatrabecular lymphoid aggregates (arrow) and increased interstitial lymphocytes; lymphocytes make up approximately 10% of the marrow cellularity.

See notes

H&E

CD20

CD3

CD20

See notes

The lymphoid aggregates and interstitial lymphocytes are mainly CD20+ B cells (CD20 stain) reveals a linear distribution of some of the B cells (arrow), suggesting localization within bone marrow sinusoids

CBC & Peripheral Smear

CBC
WBC: 3,800/mm3 Hematocrit: 20.3% Platelets: 154,000/ mm3

Differential (reference range)

63% neutrophils (40-70) 28% lymphocytes (22-44) 7% monocytes (4-11) 2% eosinophils (0-8) Absolute neutrophil, lymphocyte and monocyte counts all within normal limits

A peripheral-blood smear shows occasional mildly enlarged lymphocytes with slightly open chromatin, small nucleoli, and moderately abundant pale basophilic cytoplasm with noncircumferential villous cytoplasmic projections (arrow).

Peripheral Blood Flow Cytometry

Light scatter analysis 21% lymphocytes 11% monocytes 62% granulocytes

Lymphocyte gate 33% CD19+CD43- B cells 62% CD19-CD43+ T cells

See notes

Peripheral Blood Flow Cytometry

See notes

Peripheral Blood Flow Cytometry

Kappa-light chain restricted B-cell population dimly co-expressing CD23, negative for CD5 and CD10 Findings consistent with peripheral blood involvement by B-cell lymphoma

See notes

B-Cell Lymphomas That Lack CD5, CD10 and CD23

Splenic marginal zone lymphoma Lymphoplasmacytic lymphoma (Waldenstrms macroglobulinemia) Hairy cell leukemia B-cell lymphoma with leukemic dissemination lacking an antigen that is usually expressed
CD10- follicular lymphoma CD5- mantle cell lymphoma

The diagnostic procedure

3 cm

On gross examination of the cut surface of the spleen , there is a relative prominence of the white pulp, with nodules that range in size from 0.1 to 0.3 cm in diameter.

CD20

spleen

See notes

white pulp

On microscopical examination, the white-pulp nodules are expanded and replaced by an infiltrate of monomorphous, small lymphocytes with irregular nuclei and scant-to-moderately-abundant cytoplasm

red pulp

See notes

H&E

CD20

CD21

Ki67

See notes

Clusters of similar cells were also present in the red pulp. Immunohistochemical stains show that the atypical cells are CD20+ B cells . They strongly coexpressed IgM heavy chain, but not IgD, with immunoglobulin kappa light chain restriction by in situ hybridization (not shown).

kappa

lambda

IgM

IgD

See notes

hilar LN

See notes

Summary of Pathology
Bone marrow
Involvement by small B-cell lymphoma (non-paratrabecular lymphoid aggregates; interstitial and intrasinusoidal infiltration)

Peripheral blood
Small population of circulating villous lymphocytes

Spleen (2.4 kg)

Small IgMk+, IgD-/+ B-lymphocytes with moderately abundant cytoplasm replacing reactive follicles of white pulp, within sinusoids of red pulp; involvement of hilar lymph node Cytogenetics: 46,XX[8]; no 7q31 loss seen by FISH (100 nuclei)

Flow cytometry (blood, bone marrow, spleen)

CD5-, CD10-, CD23-/+ clonal B-cell population

Splenic Marginal Zone Lymphoma

Postulated cell of origin: post-germinal center B-cell of the splenic marginal zone Recent studies suggest greater heterogeneity in terms of histology, immunophenotype, genetics and cell of origin
Unmutated IGHV regions Associated with loss of 7q22-32 More aggressive clinical course No association with CD38 positivity Mutated IGHV regions Absence of 7q loss IgD negative Selective use of IGHV regions in V-D-J gene rearrangement Common antigen (? autoantigen) responsible for preferential VH gene usage, leading to clonal expansion and transformation
Algara P et al. Blood 2002 Stamatopoulos K et al. Mol Med 2004 Papadaki et al. Am J Surg Pathol 2007

Coagulation Abnormalities
PTT: 63.9 seconds (normal 22.1-34.0)
Presence of lupus anticoagulant suspected on screening assay (PTT-LA), confirmed by second assay (hexagonal phase) PTT-related factors
Factor VIII: >42% Factor XII: >14% Factor IX: 42% Factor XI: 38%

Not accurately quantified due to lupus anticoagulant interference

Presumably false underestimates due to lupus anticoagulant interference

Coagulation Abnormalities
PTT: 63.9 seconds (normal 22.1-34.0)
Presence of lupus anticoagulant suspected on screening assay (PTT-LA), confirmed by second assay (hexagonal phase) PTT-related factors
Factor VIII: >42% Factor XII: >14% Factor IX: 42% Factor XI: 38%

Not accurately quantified due to lupus anticoagulant interference

Presumably false underestimates due to lupus anticoagulant interference

Anticardiolipin antibodies
IgM: >150 MPL (normal 0-15) IgG: 5.2 GPL (normal 0-15)

Work-Up of Prolonged PTT

Rule-out Heparin (e.g. with heparinase)
1:1 mix with normal plasma

Re-measure PTT at 0 hours and after incubation

PTT corrects into normal range

PTT remains prolonged

PTT corrects initially then prolongs after incubation

Factor Deficiency

Lupus Anticoagulant

Factor Inhibitor
Van Cott and Laposata. In Henry JB (20th ed.), 2001

Coagulation Work-Up
PTT mixing study Initial PTT: 63.9 seconds (normal 22.1-34.0); no change after heparinase 1:1 mix with normal plasma: 49.0 seconds After 2 hour incubation: 58.1 seconds
Consistent with a lupus anticoagulant

Factor VIII mixing study (a mini-Bethesda assay) Factor VIII activity measured after 1:1 mix of patient and normal plasma at time zero and after 2 hour incubation No apparent decrease in factor VIII activity at 2 hours
Factor VIII inhibitor excluded

Pathogenesis of SMZL Associated Antibodies

Direct
IgM paraprotein itself has autoimmune activity Cases of SMZL frequently use V genes that encode autoantibodies
Also a feature of normal marginal zone B cells of spleen

Indirect
T-independent antigens promote clonal expansion of B cells through repeated activation signals Auto-reactive T-cell clones induce B-cell oligoclonal responses
Sawamura et al. Ann Hematol 1994 Papadaki et al. Am J Surg Pathol 2007 Ziakas PD. Leuk Lympoma 2006

Discussion of Management
Jeremy S. Abramson, M.D.

Splenic Marginal Zone Lymphoma

Clinical features
Median age 60s HCV+ in as many as 17% Autoimmune phenomena 10-20% B symptoms 20-25% Spleen 100% Peripheral blood 57-65% Bone marrow >90% (perhaps 100%) Liver 15-30% Systemic adenopathy 15-35% (Abdominal > Peripheral> Mediastinal) Anemia 30-65% Thrombocytopenia 15-25% Neutropenia 5% Absolute lymphocytosis 58% LDH 28-43% Monoclonal gammopathy 10-46%

Site

Labs

Troussard, et al. BJH 1996. Berger, et al. Blood 2000. Chacon, et al. Blood 2002. Thieblemont, et al. Clin Lymph 2002. Parry-Jones, et al. BJH 2003. Iannitto, et al. Cancer 2004. Arcaini, et al. Blood 2006.

Immunologic Abnormalities in SMZL

Autoimmune hemolytic anemia- warm (IgG) and cold (IgM) Immune thrombocytopenic purpura (ITP): IgG mediated Evans syndrome IgM anticardiolipin antibody and/or lupus anticoagulant Antibodies to parietal cells, intrinsic factor, thyroid peroxidase, thyroglobulin, and adrenal cortex Gradual resolution of autoantibodies post-treatment is common. May not be complete.

Ciaudo, et al. Am J Hem 1991. Murakami, et al. Am J Hem 1997. Chacon, et al. Blood 2002. Martin, et al. Leuk Lymph 2006. Ziakas, et al. Leuk Lymph 2006.

Splenic Marginal Zone Lymphoma

Outcome
Median overall survival 9 - 13 years 5 year overall survival 65-72% Lifetime transformation risk 10%

Adverse risk factors

Anemia, Increased LDH, hypoalbuminemia, lymphocytosis, and involvement of non-hematopoietic sites

Indications for Treatment

Symptomatic splenomegaly Cytopenias Autoimmune complications B symptoms
Troussard, et al. BJH 1996. Camacho, et al. AJSP 2001. Chacon, et al. Blood 2002. Arcaini, et al. Blood 2006.

Treatment Options
Observation Splenectomy Radiotherapy Chemotherapy Rituximab HCV therapy*

Treatment Options
Splenectomy
Resolution of cytopenias, paraproteinemia, and symptoms Reduction or resolution of autoantibodies Median TTP of 3-7 years Regression of marrow disease does not occur

Chemotherapy
Alkylating agents Nucleoside analogues Combination chemotherapy

Rituximab
ORR 88-100%, 43-88% CR/CRu Median time to response: 3 weeks Median FFS 2-3 years
Mulligan, et al. BJH 1991 Chacon, et al. Blood 2002. Thieblemont, et al. Clin Lymph 2002. Tsimberidou, et al. Cancer 2006. Franco, et al. Cancer 2001. Murakami, et al. Am J Hem 1997. Kalpadakis, et al. Hem Oncol 2007. Bennett, et al. Haematologica 2005.

Follow-up
Dr. Abramson

Labs Post Splenectomy

Time
HCT (%) PLT WBC Retic (%) T. Bili LDH Haptoglobin

Pre-op
19.5 130 3.2 9.2 3.6 1574 <6

20 days post-op
33.6 667 7.0 4.4 0.4 463 <6

53 days post-op
37.5 569 12.1 Not performed 0.3 302 <6

Labs Post Splenectomy

Time Pre-op 20 days post-op 53 days post-op

IgM (g/dL) IgM kappa MComponent (g/dL)

PTT (seconds) Lupus Anticoagulant IgM Anticardiolipin Antibody (MPL) Hepatitis A IgM

270 0.12
63.9

64 0.04
35.3

Not performed Not performed

30.7

Positive >150.0 Reactive

Positive >150.0 Non-reactive

Positive 114.9 Non-reactive