
Lymphoma: B cell and plasma cell neoplasms
Last revised 27 May 2008
Last major update July 2004
Copyright (c) 2001-2008 PathologyOutlines.com, Inc.
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Lymph nodes: normal development, normal histology, molecular analysis, grossing lymph nodes, features to report
Non-Hodgkin’s lymphoma: general, classification, cytogenetics, staging, morphologic clues, hemophagocytic syndrome, chemotherapeutic atypia
B cell disorders: general, WHO classification
B cell lymphoma subtypes: Burkitt, Burkitt-like, CLL/SLL, diffuse large B cell, follicular, hairy cell leukemia, intravascular large B cell, lymphomatoid granulomatosis, lymphoplasmacytic, mantle cell, marginal zone-general, marginal zone-MALT, marginal zone-nodal, mediastinal (thymic), plasmablastic, pre B lymphoblastic leukemia/lymphoma, primary cutaneous B cell, primary cutaneous diffuse large B cell, primary effusion, prolymphocytic leukemia, pyothorax associated, splenic marginal zone, splenic lymphoma with villous lymphocytes
Plasma cell neoplasms: general, myeloma, plasmacytoma, heavy chain disease, primary amyloidosis, MGUS, cryoglobulinemia
Go to Lymphomas: non B cell
(T/NK cell disorders, Hodgkin Lymphoma, Post-transplantation/other, AIDS associated lymphoproliferative disorders, Other)
American Journal of Clinical Pathology (AJCP), Jan 1997 to Apr 2002 (no photos)
American Journal of Surgical Pathology (AJSP), Jan 1999 to July 2004
Archives of Pathology and Laboratory Medicine (Archives), Jan 1999 to July 2004
Human Pathology (Hum Path), Jan 2000 to June 2004
Modern Pathology (Mod Path), Jan 2001 to July 2004
Kjeldsberg, CR: Practical Diagnosis of Hematologic Disorders (3rd Edition); ASCP Press, 2000
Sternberg, S: Diagnostic Surgical Pathology (4th Ed); Lippincott Williams & Wilkins, 2004, 728-45
Please refer to these primary references for more detailed discussions
Normal lymphoid cells undergo rearrangements within their antigen receptor genes, causing specificity for the immunoglobulin or T cell receptor that they produce
Monoclonal proliferations are presumed to be neoplastic; polyclonal populations are not
Lymphoid stem cell: TdT+, CD34+, HLA-DR+, then develops along B or T cell pathway
B cells
Develop from stem cells of yolk sac, fetal liver, spleen and bone marrow
In intersinusoidal bone marrow, hematopoeitic stem cells mature to early lymphoid progenitors, the pro-B, pre-B stages
Developmental stages are in close contact with slender CD10+ stromal cells or their extensions, which allows tightly regulated signaling (J Pathol 2005;205:311)
B cells express surface immunoglobulin (Ig), composed of 2 heavy (H) and 2 light (L) chains (either kappa or gamma)
B cell antigen receptor loci may have 4 types of modification - recombination of variable, diversity and joining regions (VDJ); somatic hypermutation of V segments; immunoglobulin heavy chain gene class switching; and receptor editing
Defects may cause chromosomal translocations
1-diagram of IgH gene rearrangement
References: Archives 2003;127:1148
Early B cell precursor is TdT+, CD34+, HLA-DR+, then undergoes heavy (H) chain rearrangement and adds CD19, then adds CD10, then adds IgM heavy chain; then adds light (L) chain rearrangement and adds cytoplasmic IgM with heavy and light chains, then B cells express IgM and IgD with the same binding site, then adds CD20 (now called preB cell); then adds surface Ig, then adds CD21 and CD22 and drops TdT (now called B cell)
If B cell encounters an antigen that interacts with its variable region, it becomes a plasma cell
Precursor B cells contain immunoglobulin related components but not immunoglobulin; express CD179a and CD179b (precursor to light chains) as part of their pre-B cell receptor, which disappears when replaced with conventional light chains
B cells express surface immunoglobulin, consisting of heavy chain and kappa or lambda light chains; immunoglobulin is associated with CD79a/CD79b complex to form a B cell antigen receptor complex
IgH (heavy chain of immunoglobulin): 14q32; variable portion coded by VDJ regions
IgL (light chain of immunoglobulin): kappa on 2p11, lambda on 22q11; no diversity region is present
Heavy chain isotype switch: determines if immunoglobulin is IgM, IgD, IgG1-4, IgA1-2 or IgE (9 constant regions); mediated by switch genes
B cell lymphomas: clonal light chain rearrangement is usually specific for the presence of a B cell neoplasm
Develop from bone marrow, become prothymocytes, then migrate to thymus gland, where self-recognizing T cells are eliminated
T cell receptors (TCR) are either alpha/beta (95%) or gamma/delta (5%) heterodimers
Precursor cell is TdT+, CD34+, HLA-DR+, then drops HLA-DR, then adds CD2, CD5, CD7 (early thymocyte) while undergoing gamma/beta chain rearrangement, then adds CD1 and drops CD34, now a common thymocyte, then undergoes beta/alpha chain rearrangement and adds CD4 and CD8, then splits into helper or cytotoxic T cell, without TdT, CD1, CD5 and CD7; has CD2, CD3, CD4 (helper) or CD8 (cytotoxic)
T alpha and delta are on 14q11; T beta is on 7q34; T gamma is on 7p15 (note: there are only 10 V regions, so a polyclonal population of cells can appear oligoclonal)
90% of peripheral T-cell lymphomas have rearrangements of T-alpha, beta and gamma, including all cases of mycosis fungoides and Sezary syndrome
T cell lymphomas: no distinct marker of clonality, but cells may express an abnormal immunophenotype
Note: T cell clonality is seen in AIDS and congenital immunodeficiency syndromes, but does NOT indicate malignancy
Note: rarely a clonal band may comigrate with the germline band; solution - use 2-3 restriction enzymes (HindIII, EcoRI, BamHI)
Note: T cells and NK cells arise from common progenitor that expresses CD3 epsilon and cannot develop into B cells
NK cells
Distinct group of non-T, non-B lymphocytes; large granular lymphocyte morphology on Wright-Giemsa stains; capable of lysing certain target cells without prior activation or major histocompatibility complex restriction; believed important in defense against viral and bacterial infections and cancers as well as immunomodulation and regulation of hematopoiesis; comprise 5-20% of peripheral blood lymphocytes
Positive stains: CD56 (adhesion molecule), CD57 (unknown function), CD16
(low affinity IgG Fc receptor / FC
RIII that is responsible for antibody
dependent cellular cytotoxicity in NK cells and also expressed on neutrophils
and monocyte subset); also cytoplasmic (not surface) CD3, CD2, CD7, CD8, perforin,
granzyme B, TIA-1
>90% are CD16+/CD56+
Centroblasts: large non-cleaved follicular center cells with moderate amounts of basophilic cytoplasm, round nuclei, open chromatin, multiple peripheral nucleoli
Centrocytes: small cleaved follicular center cells with scant cytoplasm
Leukemia: lymphoid neoplasms that present with widespread bone marrow involvement and large numbers of tumor cells in the peripheral blood
Mantle zone: Small unchallenged B cells surrounding pale staining germinal centers
Marginal zone: light zone surrounding follicles; contain post-follicular memory B cells derived after stimulation of recirculating cells from T cell dependent antigen
Germinal center: strong dense bcl6 expression and CD10 expression
TdT: terminal deoxynucleotidyl transferase; marker for premature B and T cells
Note: antigen stimulated B cells with the capacity to differentiate toward plasma cells express MUM1/IRF4 and CD138
Fluorescent in-situ hybridization (FISH)
To identify and count chromosomes / parts of chromosomes,
Northern blot
To detect mRNA
Polymerase chain reaction (PCR)
10,000 times more sensitive than Southern blot
Clones appear as 1-2 bands (if 1 or 2 alleles were rearranged); polyclonal cells appear as a smear since no amplification usually occurs due to wide separation of V and J regions
Faster than Southern blot and minimal tissue required
Helpful for determining clonality of lymphoid aggregates in bone marrow biopsies (Archives 2000;124:511)
Procedure: add sample DNA, 4 nucleotides, buffer with magnesium, primers and Taq DNA polymerase to test tube; use PCR cycler with 3 reactions (#1 - denature double stranded DNA, #2 - allow annealing of primers, #3 - allow DNA polymerase activity to extend primer); multiple cycles allow exponential expansion of amount of DNA; analyze products by gel electrophoresis, then stain gel with ethidium bromide OR transfer gel to nylon membrane by Southern blotting and hybridize membrane with labeled probe
False negatives due to imperfect consensus primers, inability to detect partial DJ rearrangements; mutations of Ig genes that prevent annealing of primers; lymphomas that arise from B cell precursors prior to rearrangement
PCR + temperature gradient gel electrophoresis
To analyze T cell receptor gene rearrangement (Archives 2001;125:202)
Reverse transcriptase PCR (RT-PCR)
To detect chimeric fusion mRNA transcripts translocations
Southern blot
Detects DNA via restriction endonucleases, electrophoresis and probes
For lymphoma, detects alterations of DNA restriction fragment length from rearrangement of immunoglobulin and T cell receptor genes
Rearrangement forms a distinct band if only 1% of total cells are affected, so very sensitive; only a single germline band is present if polyclonal or not clonal, image
Difficult and slow to perform
Procedure: extract DNA, cut into small fragments with restriction enzymes (EcoRI, HindIII, BamHI); run on electrophoretic gel (agarose); apply nylon membrane (blot) over gel to transfer DNA fragments onto membrane; denature the probe DNA and DNA fixed to the blot to allow hybridization and add probe DNA; detect the probe (xray if probe radioactive); usually analyze IgH and TCR-beta for clonality
Theory of molecular analysis
B cells express surface immunoglobulin (Ig), composed of 2 heavy (H) and 2 light (L) chains (either kappa or gamma)
T cell receptors (TCR) are either alpha/beta (95%) or gamma/delta (5%) heterodimers
Ig and TCR germline configuration contains Variable, Diversity, Joining and Constant region segments, IgH; TCR
Early in normal lymphocyte differentiation within the bone marrow, the antigen receptor genes undergo recombination to create a variable region, image, containing an antigen combining site and a constant region. Diversity occurs through recombination of segments plus imprecise V-D-J joining
Terminal deoxytransferase (TdT) adds or removes nucleotides, causing even more diversity
Point mutations in V and J regions occurs commonly within Ig genes, adding to diversity
An estimated 100 million different Ig and TCRs exist
Rearrangement forms a distinct band if multiple cells have the same rearrangement pattern, indicating clonality
80% of B or T cell lymphomas with characteristic immunologic and clinical features have clonal IgH or TCR-gamma rearrangement by PCR (Mod Path 2000;13:1269); 10% of B cell and T cell lymphomas have both clonal IgH and TCR-gamma rearrangement
DD of “clonality”: non-neoplastic tissue may be clonal, perhaps due to autoimmune diseases for B cell disorders or granulomatous diseases for T cell disorders; tissue with a small number of polyclonal B cells (skin, GI) may cause a pseudoclonal PCR profile; best to do multiple PCR and look for same rearranged band in every experiment
References: Archives 1999;123:1189
Best to obtain fresh and intact
Handle under sterile conditions for microbiology (depending on clinical history)
Use scrapes / cell suspension for flow cytometry, cytogenetics, molecular gene rearrangement studies, FISH
Obtain imprints for Wright stain or immunocytochemistry
Snap-freezing is best for research, some immunohistochemistry, future molecular studies
B5 (mercury containing fixative) provides best morphologic details
Formalin fixation is best for PCR
Use thin (2 mm) slices for proper fixation; cut perpendicular to long axis if possible
Include extranodal fat (infiltration implies malignant)
Clinical history (prior diagnoses of lymphoma, presence of lymphadenopathy or organomegaly, hematologic findings, constitutional symptoms, HIV status, prior immune abnormality, autoimmune disorders, other relevant serology, other related conditions such as H. pylori infection)
Anatomic site
Tumor type(s) (WHO classification), grade (if relevant)
Focal or complete involvement of lymph node or other structures
Specimen inadequacy
Results of ancillary studies
References: Hum Path 2002;33:1064; Mod Path 2004;17:131
Non-Hodgkin Lymphoma
Non-Hodgkin lymphoma - general
Clonal lymphoproliferative disorder
Increasing incidence over past 40 years for unknown reasons
50,000 new cases in US per year, many HIV related
Heterogenous types of neoplasms; diagnosis of “NHL” gives far less information than type of NHL
Putative cell of origin known for most B cell NHLs but not most T cell NHLs
Often associated with a cytogenetic translocation that puts a proto-oncogene or apoptotic gene next to a gene that is constitutively active in lymphocytes
80% are B cell, NK are rare
All are monoclonal, as determined by antigen receptor gene rearrangement (immunoglobulin or T cell receptor)
Characteristic patterns of tissue involvement occur, such as follicular lymphomas in B cell areas, T cell lymphomas in paracortical zones
Most tumors are widely disseminated at diagnosis, requiring systemic therapy for cure; thus, staging is not as important as in Hodgkin lymphoma
In adults, most common subtypes are follicular lymphoma, diffuse large B cell lymphoma, CLL/SLL, multiple myeloma
Usually HER2 negative (Archives 2002;126:574)
Prognostic factors: see particular tumors; cyclin D3 overexpression identifies patients with indolent B cell lymphomas but adverse clinical features and poorer survival (AJCP 2001;115:404)
Risk factors: immunodeficiency (primary or secondary), autoimmune disorders (Sjogren’s, rheumatoid arthritis, Hashimoto’s thyroiditis), viruses (HIV, ATLV, KSHV/HHV8, HTLV-1), Helicobacter pylori infection, radiation, chemotherapy
Compared to Hodgkin lymphoma: more often extranodal, more often involve peripheral blood, bone marrow, GI, skin or CNS, more often disseminated, present with B symptoms less often (20% vs. 40%), less often mediastinal involvement (except for lymphoblastic and mediastinal large B cell lymphoma)
International Prognostic Index (IPI): poorer prognosis - age at diagnosis >60 years, presence of B symptoms, performance status 2-4 vs. 0-1, elevated serum LDH, more than 1 nodal or extranodal sites of disease, advanced vs. localized disease
Treatment: addition of rituximab (anti-CD20 antibody) to most regimens leads to improved survival
DD: florid immunoblastic proliferations seen in infectious mononucleosis or other viral infections, particularly in children; autoimmune lymphoproliferative syndromes in patients in Fas or FasL deficiency may develop giant lymphadenopathy resembling EBV+ post-transplant lymphoproliferative disease (AJSP 1999;23:829)
Childhood NHL
Most common subtypes are Burkitt or Burkitt like, lymphoblastic leukemia/lymphoma, large cell lymphoma
Follicular and marginal zone lymphoma are uncommon
Usually extranodal, aggressive, often leukemic; better survival than adults
Fine needle aspiration
Sensitivity and specificity increase with flow cytometry; good in most lymphomas except marginal zone lymphoma and Hodgkin lymphoma
References: Archives 2000;124:1792
Rappaport classification: 1956, revised 1966
Lukes and Collins classification, 1974
Working Formulation: 1982; tumors classified as low, intermediate or high grade; nodular vs. diffuse; small, large or mixed tumor cell size
Kiel classification: European system used in 1980-1990’s
REAL (Revised European American Lymphoma) / World Health Organization (WHO): integrates clinical, morphologic, immunohistochemical and molecular characteristics
Includes NHL, lymphocytic leukemias, plasma cell neoplasms; excludes histiocytic neoplasms
Tumors are not classified as low grade / high grade since one entity could have both types
High concordance under REAL between diagnoses in community hospital and academia; discordances seen for diffuse large cell lymphoma vs. Burkitt-like lymphoma, marginal zone lymphoma vs. another subtype, follicle center lymphoma grade II vs. grade III, Hodgkin lymphoma-nodular sclerosis vs. mixed cellularity (AJCP 2001;115:650)
Relatively common translocations (more complete lists are listed with each tumor)
t(1;14)(p22;q32): bcl-10 and IgH; MALT lymphoma (% unknown)
t(1;14)(p32;q11): SCL (tal-1) and T cell receptor delta/alpha; precursor T acute lymphoblastic leukemia (15-30%)
t(1;14)(q21;q32); bcl-9 and IgH; preB ALL, mantle cell lymphoma
t(1;19)(q23;p13): PBX1 and E2A; precursor B acute lymphoblastic leukemia (30%)
t(2;5)(p23;q35): ALK and NPM; anaplastic large cell lymphoma, T/NK subtypes (40-70%)
t(2;8)(p12;q24): Ig Kappa and c-myc; Burkitt lymphoma (15%)
t(2;18)(p12;q21): Ig Kappa and bcl2; follicular lymphoma (<5%)
Trisomy 3: MALT lymphoma (% unknown)
t(3;14)(q27;q32): bcl6 and IgH; diffuse large B cell lymphoma (30%), follicular lymphoma (10%)
t(4;11)(q21;q23): AF4 and MLL; precursor B acute lymphoblastic leukemia (10%)
t(4;14)(p16.3;q32): FGFR3/mmset and IgH; multiple myeloma (25-30%)
t(5;14): preB ALL and peripheral eosinophilia; IL3 gene and IgH
t(6;14)(p25;q32) - mum/irf4 and IgH; multiple myeloma
7q isochromosome: protein unknown, hepatosplenic gamma/delta lymphoma (% unknown)
Trisomy 8: protein unknown, hepatosplenic gamma/delta lymphoma (% unknown)
t(8;13)(p11;q11-12): FGFR1 and ZNF 198; T cell lymphoblastic with eosinophilia (% unknown)
t(8;14)(q24;q32): c-myc and IgH; Burkitt lymphoma (75%), acute lymphocytic leukemia-type L3 (6%)
t(8;21): ETO gene and AML1 gene; AML-M2
t(8;22)(q24;q11): c-myc and Ig Lambda; Burkitt lymphoma (10%)
9p amplification: REL; primary mediastinal large B cell lymphoma (% unknown)
t(9;14)(p13;q32): PAX5 and IgH; lymphoplasmacytic lymphoma (% unknown)
t(9;22)(q34;q11): c-abl and bcr (Philadelphia chromosome); precursor B acute lymphoblastic leukemia
(5% of children, 25% of adults), chronic myelogenous leukemia (100%)
t(10;14)(q24;q11): HOX11 and T cell receptor delta/alpha; precursor T acute lymphoblastic leukemia (7%)
deletion of 11q23: CLL (10-20%)
t(11;14)(q13;q32): bcl-1/PRAD1 and IgH; mantle cell lymphoma (90%), B cell prolymphocytic leukemia (20%), myeloma (3%)
t(11;14): T-ALL; rhombotin 1/2 genes and IgH
t(11;18)(q21;q21): API2 and MLT; MALT lymphoma (50%)
Trisomy 12: protein unknown, B chronic lymphocytic leukemia (30%)
t(12;21)(p13;q22): ETV6-CBFA2 (TEL and AML1); Precursor B acute lymphoblastic leukemia (20%)
deletion 13q14: protein unknown; B cell CLL (25-50%)
inversion 14(q11;q32) or other #14 translocations: T cell prolymphocytic leukemia (75%)
t(14;15)(q32;q11-13); IgH and bcl-8; diffuse large B cell lymphoma (3%)
t(14;16)(q32;q23); IgH and c-maf; multiple myeloma
t(14;18)(q32;q21): IgH and bcl2; follicular lymphoma (90%), diffuse large B cell lymphoma (30%), image
t(14;19)(q32;q13): IgH and bcl-3; B cell CLL
t(15;17): retinoic acid receptor and PML gene; acute prolymphocytic leukemia, M3 (most)
t(16;22);(q23;q11): cmaf and Ig lambda; multiple myeloma
Trisomy 18: common in marginal zone lymphoma, MALT type
Staging of Non-Hodgkin lymphomas
I: one anatomic region or two contiguous regions on same side of diaphragm
II: two or more anatomic regions or noncontiguous regions on same side of diaphragm
III: both sides of diaphragm, but lymph nodes or spleen only
IV: any lymph node region plus liver, lung or bone marrow
National Cancer Institute Modified Staging for Intermediate and High Grade Lymphomas
I: Localized disease (nodal or extranodal)
II: Two or more nodal sites or a localized extranodal site plus draining sites plus either performance status <=70, B symptoms, any mass > 10 cm, serum LDH > 500, 3 or more extranodal sites of disease
III: Stage II and any poor prognostic factor
Morphologic clues to diagnosis
Small cells, round: CLL/SLL
Small / intermediate cells with atypia: Mantle cell lymphoma (irregular), MALT lymphoma (monocytoid)
Intermediate cells: follicular lymphoma (cleaved), Burkitt lymphoma (non-cleaved, prominent nucleoli), lymphoblastic lymphoma (fine chromatin)
Large cells: diffuse large B cell lymphoma, mycosis fungoides or Sezary syndrome (cerebriform), anaplastic large cell lymphoma (pleomorphic)
May be associated with non-Hodgkin lymphoma (most common: diffuse large B cell, T cell, NK)
Systemic presentation (fever, splenomegaly, jaundice), survival < 2 years, mild interstitial lymphoid infiltrate of bone marrow at presentation (AJSP 2001;25:865)
Treatment: cytotoxic chemotherapy if EBV related vs. treatment of infection in non-EBV infection
Gross: often no pronounced tumor mass
Micro: hemophagocytosis (phagocytosis by macrophages of white blood cells, red blood cells, platelets and precursors); in bone marrow, mild interstitial lymphoid infiltrate at presentation
Positive stains: depends on tumor type
Molecular: occasional HHV6 positive by PCR
Chemotherapeutic agents, particularly alkylating agents, can induce bizarre epithelial atypia
Observed in lower respiratory tract and sinonasal tract
Includes patients treated for leukemia, myeloma
Micro: striking nuclear enlargement, hyperchromasia and pleomorphism
DD: dysplasia, particularly in frozen sections (AJSP 2001;25:652)
B cell disorders
Almost always surface immunoglobulin light chain positive
Low grade tumors may have extensive sarcoid-like granulomas (Archives 2000;124:152)
B cell non-Hodgkin lymphomas rarely are CD2+, but behavior is similar to CD2- tumors; must differentiate from composite B, T cell lymphomas (AJCP 2001;115:396)
Micro images: granulomas in low grade lymphoma
Post-rituximab (anti-CD20 antibody)
Bone marrow specimens may contain aggregates of T cells that suggest relapse; so ordering immunostains is important (AJCP 1999;112:844)
CD79a and Pax-5 should be used as the first-line B lineage-specific markers for CD20 negative mature B-cell lymphomas; if negative, OCT.2 or BOB.1 may be useful (AJCP 2006;126:534)
B cell disorders - bone marrow biopsy
Overall incidence of marrow involvement at diagnosis of 40-55% in adults, varies from 20% (MALT lymphoma) to 80% (splenic marginal zone / mantle cell lymphoma)
Usually less than half of marrow is involved, particularly in SLL and follicular center cell lymphoma
Patterns of involvement are diffuse, focal paratrabecular, focal nonparatrabecular, interstitital intrasinusoidal
Rare patterns are intravascular or recapitulating nodal involvement by neoplasm
Focal paratrabecular: suggests follicular center cell lymphoma
Interstitial pattern: preservers marrow architecture but infiltrates interstitium of marrow; may appear normal on low power
Intrasinusoidal pattern: difficult to identify without special stains (CD20)
Predominant cell type in marrow usually is similar to that in lymph node, particularly for Burkitt’s lymphoma and T cell lymphoma; may be discordant for follicular lymphoma (marrow-small cleaved cells; lymph node-large cells or mixture of large and small cells)
Discordance of marrow and nodal lesions may be due to different clones, a benign lesion at one site or post-antibody therapy
Granulomas are common and non-specific
Note: presence of small, incidental, clonal B cell populations in peripheral blood or marrow often is NOT associated with subsequent lymphoma, at least within a few years (AJCP 2004;122:588)
Immunohistochemistry: important to define cell type involved, extent of involvement, post-treatment residual disease and whether it has same lineage as pre-treatment disease