Chapter 12
The Role of Fine Needle Aspiration Biopsy in the Diagnosis
and Management of Hematopoietic Neoplasms
Rana S. Hoda
FINE NEEDLE ASPIRATION
Introduction
Fine needle aspiration (FNA) is a well-established minimally
invasive technique for the cytologic evaluation of suspicious
mass lesions involving lymph nodes and other organs. The
2008 WHO classification of tumors of hematopoietic and
lymphoid tissues utilizes a multiparametric approach based
on cytomorphologic, immunophenotypic, and cytogenetic
features to classify these tumors (1). This approach can be
applied to FNA cytologic specimens (2–8), of both nodal
and extranodal sites. About 25% to 40% of lymphoid neoplasms arise outside of the lymph node system, and dissemination of nodal neoplasms may occur to almost any
anatomical site, presenting as an extranodal mass (8). FNA
in conjunction with flow cytometry (FCM) has been shown to
be useful for the diagnosis and subclassification of lymphoma
(2–4,8–10).
The use of FNA for in the initial workup of patients with
hematolymphoid neoplasms, particularly for patients who present with lymphadenopathy, remains underutilized (Table 12.1).
Lymphoma constitutes approximately 10% of cases diagnosed
by FNA (2,7,9–11).
Indications of FNA of Lymph Nodes
The utility of FNA in lymphadenopathy varies with the clinical situation (Table 12.2). The majority of cases of lymphadenopathy are benign or reactive, and FNA can be particularly
advantageous in the diagnosis and appropriate management
thereof (12).
Accuracy of Reporting FNA of Lymph Nodes
Diagnostic accuracy of FNA for high-grade non-Hodgkin
lymphoma (NHL) and Hodgkin lymphoma (HL) (with the
exception of lymphocytic predominant variant) is high (70%
to 90%) (2,13). However, FNA has significant limitations in
Table 12.1
REASONS FOR UNDERUTILIZATION OF
FNA FOR HEMATOLYMPHOID LESIONS
• Relative ease of excisional biopsy
• Ignorance of terminologies and classification
• Limited experience
• Limited availability of cytopathologists
• Nonavailability of ancillary tests
• Lack of histologic architecture
• Morphologic difficulty in distinguishing reactive and low-grade
lymphomas
the assessment of low-grade B-cell lymphomas with a relatively low diagnostic accuracy (67%), inherent to the loss of
architecture (14). False-negative rate of FNA for lymphoid
lesions ranges from 5% to 15%, with low-grade lymphomas
accounting for a significant percentage of the false-negative
or indeterminate cases (14,15). The major strength of FNA
in the workup of hematopoietic and lymphoid lesions is its
high specificity (85% to 100%) even in extranodal sites. The
diagnostic sensitivity of FNA in the initial workup of hematolymphoid lesions is enhanced when it is used in conjunction
with FCM and/or needle core biopsy (16). A close interaction
between cytopathologists and hematopathologists to coordinate the morphologic and immunophenotypic data is also
required (7,8).
Cytologic Preparations for FNA Specimens
Aspirated material for evaluation of hematolymphoid lesions
is first processed as direct smears, both air-dried and
­alcohol-fixed smears (Fig. 12.1). Air-dried smears are stained
with a Romanowsky method. In this respect, Diff-Quik (DQ)
stain, which provides better assessment of background and
cytoplasmic features, is preferred. Alcohol-fixed smears are
best stained with Papanicolaou (Pap) stain, which is useful for
assessing nuclear features. Hematoxylin and eosin (H&E) stain
can also be used for fixed smears. Residual material is rinsed
for preparation of liquid-based preparations (LBPs), such as
ThinPrep (TP [Hologic Inc., Bedford, MA]) or SurePath (SP [BD
Diagnostics, Burlington, NC]), or for the preparation of cell
block (17). LBPs are preferably not used as the sole preparation type for FNA of suspected lymphoma because of artifactual aggregation and clumping of lymphoid cells (Fig. 12.2),
but are best suited for immunocytochemistry (ICC) and molecular analyses.
FNA Technique and Triage
Superficial FNA, by palpation only, is usually performed
using thin needles, generally 22 to 27 gauge, and most often
1 to 1.5 inch in length attached to a 5 or 10 mL disposable
Table 12.2
INDICATIONS OF FNA OF LYMPH NODES
• Distinguish between benign and neoplastic lesions
• Diagnosis of infection and to obtain material for bacterial or fungal
cultures
• Confirmation of suspected metastases
• Diagnosis of hematolymphoid lesions and to obtain ancillary studies
• Document disease extent, relapse, progression, or transformation of
­hematolymphoid neoplasm
• Monitor effects of therapy in hematolymphoid neoplasms
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FIGURE 12.2. Both liquid-based techniques, TP and SP, produce artificial aggregations of
lymphocytes that may be mistaken for epithelial cells (Pap stain, TP ×60 objective).
performed by radiologists with CT guidance using a 21- and
22-gauge Chiba needle (20,21). EUS-FNA is considered the
procedure of choice for tissue sampling of abdominal and
mediastinal nodes that may be difficult to reach by percutaneous CT guidance. EUS-FNA combined with FCM has a
reported accuracy of 70% to 90% in the diagnosis of intrathoracic and intra-abdominal lymphoma (14,22,23). EUS
also has the ability to obtain biopsy for histologic evaluation
(14,23).
A cytologist should assess on-site adequacy at the time of
FNA by the air-dried DQ-stained smears and perform proper
specimen triage (24,25). When a hematolymphoid lesion is
suspected, residual and additional specimen is rinsed in either
a phosphate-buffered saline solution or RPMI (Roswell Park
Memorial Institute) cell media for FCM. When an inflammatory/infectious etiology is suspected, microbiology studies can
be obtained. Tables 12.3 to 12.5 outline the advantages and
limitations of FNA.
FIGURE 12.1. Gross appearance of various types of cytologic preparations. (Top row
from left to right) Diff-Quik (DQ)-stained direct smear (DS), Papanicolaou (Pap)-stained
DS, hematoxylin and eosin (H&E)-stained cell block section; (bottom row from left to right)
ThinPrep (TP [20 mm diameter circle]), SurePath (SP [13 mm diameter circle]), and cytospin
(CS [12 mm diameter circle]).
plastic syringe that may be placed on a Cameco aspiration gun.
Shorter gauge needles or “butterfly” needles may be used for
superficial subcutaneous lesions (18).
High-resolution ultrasound (US) technology is considered
the “gold standard” for evaluation of superficially located targets. Pathologists are currently being trained and certified
by the College of American Pathologists (CAP) in performing
US-guided FNA (19). Image guidance enables anatomic localization of the target and its relationship to adjacent tissues.
Specific features of the target including its size and shape can
be visualized.
Suspected lymphomas of deep-seated organs are sampled
by the help of radiologic techniques including computed
tomography (CT) or endoscopic ultrasound (EUS) techniques. Transthoracic and transabdominal FNA are generally
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Strategies of FNA Interpretation
Strategies for the evaluation of FNA of lymph nodes are outlined in Figure 12.3. Interpretive problems may result from
improper FNA technique, faulty smearing technique, or
lesional characteristics: (i) improper FNA technique fails to
yield smears that are sufficiently cellular; (ii) faulty smearing
Table 12.3
ADVANTAGES OF SUPERFICIAL FNA
• Simple, easy, safe, rapid
• Cost-effective
• Noninvasive
• Preserves native lymph node architecture
• Ability to sample multiple lymph nodes
• Can be combined with FCM
• Fresh and pure tumor cells obtained for molecular tests
• Easily repeated allowing serial (pretherapy and posttherapy) sampling
• Complications are rare
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Table 12.4
ADVANTAGES OF RADIOLOGICALLY
GUIDED FNA
Table 12.5
• Small or deep-seated targets can be sampled
• Nature of target (solid or cystic) can be assessed
• Reduced number of nondiagnostic biopsies, after on-site cytologic a­ ssessment
of adequacy
• Follow-up of benign tumors for growth or change at defined intervals with US
• No ionizing radiation exposure with US
• Inability to subclassify lymphoma
• Loss of important architectural patterns
• Inability to reliably distinguish RHL from low-grade lymphomas
• Inability to reliably distinguish between various types of low-grade lymphoma
• False-negative results due to
• Sampling error with inadequate sampling due to
–– Poor biopsy technique
–– Peripheral blood contamination
–– Marked nodal fibrosis
–– Partial involvement of lymph node by tumor
–– Extensive necrosis or inflammation
• Interpretive errors
–– Faulty smearing techniques
–– Paucity of diagnostic cells such as Reed-Sternberg cells
• Rare, post-FNA changes in lymph nodes that may occur and interfere with
histologic assessment
• Total infarction
• Hemorrhage
• Granulation tissue
Poorly-stained
Artifacts
Smear
LIMITATIONS OF FNAB
• Inability to subclassify lymphoma
• Loss of important architectural patterns
• Inability to reliably distinguish RHL from low-grade lymphomas
• Inability to reliably distinguish between various types of low-grade
lymphoma
• False-negative results due to
• Sampling error with inadequate sampling due toa
–– Poor biopsy technique
–– Peripheral blood contamination
–– Marked nodal fibrosis
–– Partial involvement of lymph node by tumor
–– Extensive necrosis or inflammation
• Interpretive errors
–– Faulty smearing techniques
–– Paucity of diagnostic cells such as Reed-Sternberg cells
• Rare, post-FNA changes in lymph nodes that may occur and interfere with
histologic assessment
• Total infarction
• Hemorrhage
• Granulation tissue
a
Three or more passes with sampling from different parts of the lesion or lymph node will reduce
sampling error. Sampling error may be reduced with the use of US guidance.
Acellular/paucicellular
Thick smears,
bloody smears,
“crush” artifact
FIGURE 12.3. Strategies of FNA interpretation.
Cystic squamous cell carcinoma
congenital cysts, salivary
gland lesions, stromal processes
“Nodules”
Cellular
Dispersed
Monotonous
Polymorphous
Polymorphous
Carcinoma
Melanoma
ML, follicular,
grades 1 and 2
Childhood round
cell tumors
Carcinoma
Melanoma
Nodular lymphoid hyperplasia
ML, follicular grades 2 and 3
Diffuse lymphoid hyperplasia
Hodgkin lymphoma
ML, follicular, peripheral,
T-cell lymphoma,
Carcinoma, Melanoma
Monotonous
Suspect ML
Rare events
Granulomas
Reed-Sternberg cells
Small cells
Large cells
Round nuclei, clumped chromatin
Round and/or irregular vesicular nuclei with
multiple small nucleoli-
ML, SLL/CLL
Hodgkin lymphoma, lymphocyte predominant,
Burkitt lymphoma, atrophic lymph node
ML, diffuse large B-cell
Irregular nuclei, creases/folds
Round nuclei, coarsely reticulated
chromatin with multiple nucleoli
ML, follicular, grades 1 and 2
Burkitt lymphoma
Convoluted nuclei, fine chromatin
ML, lymphoblastic
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FIGURE 12.4. Technical issues with lymph node aspirate preparation. A: A smear preparation with unevenly distributed cellular material
that is too concentrated for proper staining. B: Excessive pressure during smear preparation showing “crush” artifact (A and B, DQ stain,
DS ×20 objective).
technique can result in concentration of the cellular material, resulting in poorly stained smears (Fig. 12.4A) or excessive pressure during smear preparation may result in “crush”
artifact (Fig. 12.4B); or (iii) lesional characteristics—centrally
necrotic masses are best sampled at the periphery or it may
result in adequate sampling. Some lymphoid neoplasms are
associated with marked sclerosis, such as nodular sclerosing
Hodgkin lymphoma (NSCHL), and may yield paucicellular samples (26,27).
Histologic evaluation of hematolymphoid processes is based
on interpretation of architectural and cellular patterns that provide important clues in interpretation. Adopting the histopathologic approach to cytologic evaluation of lymph nodes enables
the cytopathologist to discern vague architectural patterns on
scanning magnification. Small cellular aggregates or dispersed
cells pattern can be identified (Fig. 12.5A and B). Lymph nodes
that display cellular patterns of an admixture of small, mediumsized, and large lymphocytes (polymorphous population) in
addition to tingible body macrophages and histiocytes are usually associated with benign or reactive conditions (Fig. 12.6A–C).
In contrast, a monotonous or monomorphous cell population
generally implies a malignant condition (Fig. 12.7A–C). Several exceptions to this rule exist including HL and lymphoma
with a high content of epithelioid histiocytes that may show
polymorphous cells (Fig. 12.7D). Monotony in a lymph node
aspirate also does not always indicate lymphoma. Carcinomas
may appear discohesive and monotonous (Fig. 12.8). Examination of cytologic features at high magnification allows further
separation into monomorphous and polymorphous lymphoid
cell populations (Fig. 12.9A and B).
Proliferative and apoptotic indices may also be used on
FNA samples for subclassification and histologic grading of
B-cell NHL (28,29). An increase in the mean Ki-67 proliferation
index has been reported with tumor aggressiveness. The Ki-67
median percentages of 15% for indolent lymphomas and 50%
for aggressive lymphomas have been reported that show good
correlation with histologic samples (Fig. 12.9C) (28). Apoptotic
index may be incorporated with other cytomorphologic criteria. An apoptotic index of more than 3.5% may indicate a highgrade lymphoma (Fig. 12.9D) (29).
FIGURE 12.5. Architectural patterns on scanning magnification on cytologic smears. A: Small nodular aggregates indicating lymphoid
follicles in reactive follicular hyperplasia. B: A dispersed cell pattern from a reactive lymph node (A and B, DQ stain, DS ×20 objective).
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FIGURE 12.6. Polymorphous lymphoid cells in a reactive lymph node FNA. A: Smear
displays small, medium-sized, and large lymphocytes in addition to tingible body
macrophages and histiocytes (A, DQ stain, DS ×40 objective). B,C: Higher magnification displays tingible body macrophages with round-to-oval nuclei, pale chromatin,
small nucleoli, and cytoplasmic cellular debris; note the mature compact chromatin
of polymorphous lymphoid cell nuclei (A, DQ stain, DS and B, Pap stain, DS ×100
objective with oil immersion).
FIGURE 12.7. Nature of lymphoid cells in lymphoma. A,B: Smear from CLL depicts a monotonous small cell population that generally
implies malignancy. Compare the size of neoplastic lymphoid cells with red blood cells (RBCs [A, DQ stain, DS and B, Pap stain, DS ×60
objective]). C: Smear from a case of DLBCL displaying monotonous population of large neoplastic lymphoid cells. Compare the nuclear
size with scattered benign lymphoid cells (DQ stain, DS ×100 objective with oil immersion). D: HL and lymphoma with a high content of
epithelioid histiocytes that may show polymorphous cells (DQ stain, DS ×60 objective).
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ANCILLARY METHODS FOR DIAGNOSIS
OF HEMATOPOIETIC AND LYMPHOID
LESIONS
The success of FNA in the diagnosis of hematolymphoid lesions
relies on adopting a multidisciplinary approach, that is, analyzing
cytomorphology, ICC, and FCM (2–8,27,30–35). Molecular studies
further enhance diagnostic sensitivity and specificity of FNA (36).
Immunochemistry
FIGURE 12.8. Occasionally, carcinomas may appear discohesive and monotonous. In this
case of metastatic urothelial carcinoma in lung, the smear shows monomorphic epithelial cells. Clinical history, review of urothelial carcinoma histology, and a cercariform cell
(11 o’clock position) provide a helpful clue to the primary malignancy (DQ stain, DS ×60
objective).
The application of ICC has enhanced the ability to diagnose and
classify lymphomas. ICC can be successfully applied to FNA specimens. ICC is preferable in those cases where FCM has a high
false-negative rate such as large cell lymphomas, HL, and metastases (37,38). An abbreviated antibody panel including leukocyte common antigen (CD45), cytokeratin (AE1/AE3), and S-100
protein can be applied initially. In suspected hematolymphoid
lesions, an expanded battery of monoclonal antibodies can be
employed including CD20, CD79a, and PAX5 (markers for B-cell
lymphoma) (Figs. 12.9C and 12.10A and B). The presence of
FIGURE 12.9. Evaluation of nuclear features. A: FNA smear from a case of DLBCL demonstrates monomorphic malignant cells. Note the
large tumor cells size (compare with RBCs), nuclear contour irregularity, and nucleoli. Numerous lymphoglandular bodies (cytoplasmic
fragments) are present close to bare nuclei. In comparison to (A), the smear in (B) is from a reactive lymph node showing polymorphous
small lymphoid cells. Note the mature chromatin. Tingible body macrophage and a mitotic figure are also seen (A and B, DQ stain, DS ×100
objective with oil immersion). C: Cells from a case of DLBCL showing a Ki-67 index of 100% (DS ×60 objective). D: Increased apoptosis
from a case of a high-grade lymphoma (Pap stain, DS ×60 objective).
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms299
FIGURE 12.10. Immunocytochemistry. A: Cytoplasmic positivity for CD45 in a case of DLBCL. B: Strong nuclear positivity for Pax5 in a
case of CLL/SLL (DS ×60 objective).
light-chain restriction may be the most useful criterion for determining B-cell malignancy and can be applied to limited cytology
sample or in cell block sections of adequate sample (31). Evaluation of antigen loss, crucial to the immunologic diagnosis of
peripheral T-cell lymphoma (PTCL), may be more difficult to perform in FNA smears than in frozen sections, because assessment
of antigen expression within specific regions of the lymph node is
not possible. In cytology, ICC can be performed on additional LBP
including TP or SP, cytospins, or cell block sections (37).
The advantages of ICC over FCM are the requirement of a
small sample, preservation of cellular morphology, use in cases
of large cell lymphomas where FCM may be falsely negative,
and ability to assess staining pattern and intensity of staining.
The disadvantage of ICC is that evaluation of double antigen
expression (e.g., CD5/CD23 or CD10/BCL2) is not possible.
Flow Cytometry
Immunophenotyping by FCM in the diagnosis of lymphoma is
performed for the detection of clonality, which distinguishes
lymphoma from reactive lymphoid hyperplasia (RLH). The utility of FNA in conjunction with FCM for definitive diagnosis of
NHL is well documented (2,4,7,9,13,30,39–42). Several studies
of FNA and FCM have used the REAL/WHO classification system for diagnosis and subclassification of NHL. Meda et al. (2)
evaluated 290 aspirates from 275 patients. A definitive diagnosis of NHL was made in 76.7% on the basis of FNA and FCM,
which resulted in a sensitivity of 95% and specificity of 85%.
Dong et al. (4) diagnosed 67% of primary and recurrent lymphomas using FNA combined with FCM. In another reported
series, all 74 cases of lymphoma on FNA were correctly diagnosed (9). In this study, correct classification was more accurate
in 53 cases that were also subjected to concurrent FCM (84%
vs. 33%). These studies indicate the importance of using FNA
Table 12.6
FALSE-NEGATIVE FLOW CYTOMETRY
• Sampling error
• Partial involvement of lymph node by disease
• Insufficient number of neoplastic cells
• Few viable cells
• Peripheral blood contamination
• Large cell lymphoma
• Primary T-cell lymphoma
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in conjunction with FCM for diagnosis and subclassification of
lymphoma.
FCM is sensitive, provides quantitative results, and can
detect a small abnormal cell population in a reactive background. FCM has the advantages of multiparametric analysis
and objective interpretation. The panel of antibodies utilized in
FCM depends on the amount of FNA material provided. The use
of an FCM panel comprising of B- and T-cell counts, kappa and
lambda, CD4/CD8 ratio, and proliferation index is effective in
detection of NHL subtypes and distinguishing them from RLH.
The ability to distinguish between monotypic and polytypic lymphoid cell population is considered a major advantage of FCM.
The overall accuracy for diagnosis and subclassification
of B-cell lymphomas by FCM performed on FNA samples is
reported to be 88.4% with a sensitivity of 86% and specificity
of 93% (39). Similar concordance (88%) has been reported for
FCM performed from excised tissue and subsequent histopathologic diagnoses (2,4,9,10). The sensitivity of FCM for T-cell
NHL is poor and reported to be 36.4% (39).
FCM may be false negative in large cell NHL and HL. However, the cellular abnormalities in these lesions are easily appreciated on FNA smears and should prompt additional diagnostic
studies. FCM may be misinterpreted as polyclonal due to a small
fraction of tumor compared to a prominent component of benign/
reactive lymphocytes such as in T-cell lymphomas. When cellularity is limited because of fibrosis, extensive necrosis, or cellular
damage, FCM results may be inconclusive (Table 12.6) (2,40).
Molecular Analyses
Molecular analyses in the diagnosis of lymphoma are performed
for the detection of genetic abnormalities such as translocations,
deletions or aneuploidy, and gene rearrangement. Sufficient DNA
and RNA may be obtained from FNA-derived material for analysis by polymerase chain reaction (PCR), or fluorescence in situ
hybridization (FISH) and nucleic acid microarrays/gene profiling
techniques. For molecular analyses, specimen can be directly
collected into RNA-preserving or extracting solutions or in PreservCyt solution (cell collection and preservation medium for TP
[Hologic Inc., Bedford, MA]) that retains both cytologic features
and RNA for assessment (36,43,44). These techniques can also
be applied to archival cytologic material (TP, alcohol-fixed or
formalin-fixed, paraffin-embedded). The results are comparable
to those in excision biopsies (36). DNA obtained from FNA samples may be analyzed by PCR for the presence of specific chromosomal translocations, viral genomes, activated or amplified
oncogenes, and gene rearrangements. For example, detection
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of t(14;18) (q32;q21) on PCR may provide evidence of a follicular lymphoma (FL) and t(11;14)(q13;q32) evidence for mantle
cell lymphoma (MCL) (36,43). In addition, PCR studies on cytology specimens may be used to determine viral load in patients
infected with HIV. The pattern of gene rearrangements obtained
on analysis of DNA from FNA cytology specimens is comparable
to that obtained on excised lymph nodes (30). In an appropriate sample, interphase FISH can exploit intact interphase nuclei
and detect structural and numerical chromosomal abnormalities (36,43). DNA microarrays can rapidly provide information
on many genes at the mRNA level. It has been possible to use
amplified RNA from small FNA samples for gene expression
profiling in combination with morphology and immunophenotyping for subtyping of diffuse large B-cell lymphoma (DLBCL).
The amount of RNA recovered from FNA has been shown to be
comparable to that from a core biopsy (36,41).
NON-HODGKIN LYMPHOMA
Cytologic subclassification of NHL, particularly of small mature
B-cell lymphomas, is controversial and underutilized (2,7,9–11).
However, FNA in combination with ancillary tests including ICC, FCM, and/or molecular analyses can be used in the
evaluation of NHL (2,4,7,9,13,27,30,36–44). Ninety percent of
NHLs are B-cell type, with more than half representing either
DLBCL or FL. Some lymphomas have characteristic cytologic
appearances, and it may be possible in these cases to render
a diagnosis of lymphoma based on cytomorphology alone, at
least in general terms. On FNA of most NHL, a monotonous
lymphoid cell population with uniform nuclear size and chromatin is evident (Figs. 12.7A–C and 12.9A). Once the monotony
of the lymphoid population is appreciated, the initial subclassification into small and large lymphoid cells requires the comparison of the suspect neoplastic lymphoid cells to typical small
mature lymphocytes, red blood cells, plasma cells (PCs), or histiocytes (Figs. 12.7A–C and 12.9A). Nuclei of histiocytes can
be used as an internal reference to assess the relative size of
the lymphoid cells and separate them into small- and large cell
types (Figs. 12.6B, C and 12.9B). Careful evaluation of nuclear
features including contour, chromatin pattern, and nucleolus
aids in further subclassifying lymphoid cells on cytology (Figs.
12.6B, C, 12.7C, and 12.9A and B) (26,27).
DIAGNOSTIC CATEGORIES OF FNA FOR
HEMATOPOIETIC LESIONS
Cytologic diagnostic categories for FNA of hematopoietic and
ymphoproliferative
lymphoid neoplasms include “atypical l­
lesion,” “atypical lymphoid infiltrate,” “suspicious for lymphoma,” “malignant lymphoma,” or “B-cell lymphoma” (the
latter, if immunophenotyping has been performed) (7,25).
­Figure 12.11 outlines an algorithmic approach to evaluating
FNA of lymph nodes for appropriate diagnostic categorization.
B-Cell Neoplasms
Lymphoblastic Lymphoma
Lymphoblastic lymphoma (LBL) is of either precursor B- or
T-cell differentiation (Table 12.7). It is discussed in the T-Cell
and Natural Killer-Cell Lymphoma section.
FIGURE 12.11. An algorithmic approach to evaluating FNA of
lymph nodes.
Enlarge Lymph Nodes
FNA
Lymphoid Cells
Non-lymphoid cells
Metastatic carcinoma
Reactive
Infectious
Lymphoma
Non-specific
Granulomatous
Hematolymphoid
Non-hematolymphoid
Non-granulomatous
Primary
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Others
Lymphocytic component
Non-Hodgkin Lymphoma
Large cells
Melanoma
Mixed cell pattern
Hodgkin Lymphoma
Recurrent
Primary
Recurrent
Small cells
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms301
Table 12.7
2008 WHO NHL, B-CELL SUBTYPES
REVIEWED IN THIS SECTION
• Precursor B-cell lymphoblastic lymphoma
• Mature B-cell neoplasms
• SLL
–– CLL/SLL
–– LPL
–– Plasmacytoma
–– Extranodal MZL of MALTtype
–– N-MZL
–– FL
–– MCL
• Aggressive B-cell lymphomas
–– DLBCL, not otherwise specified
–– PBL
–– BL
Mature B-cell Neoplasms
Chronic Lymphocytic Leukemia/
Small Lymphocytic Lymphoma
Key Cytologic Features
• Monomorphic small lymphocytes
• Regular round nuclei, clumped chromatin, small to inconspicuous nucleoli
• Scant cytoplasm
• Promyelocytes and paraimmunoblasts
Clinical Features. Chronic lymphocytic leukemia/small lympho­
cytic lymphoma (CLL/SLL) is a B-cell neoplasm that constitutes
about 5% of all NHLs. SLL is regarded as a tissue manifestation
of B-cell CLL and often involves lymph nodes. It predominantly
occurs in older adults and is more common in men. Patients
are usually asymptomatic, but anemia, hepatosplenomegaly,
and generalized lymphadenopathy may be present. Patients
also have peripheral blood and bone marrow involvement
at the time of presentation. Although CLL/SLL is an indolent
lymphoma, approximately 5% of cases can transform to a highergrade lymphoma clinically referred to as Richter syndrome or
transformation. FNA can be used to evaluate patients with a
history of CLL/SLL who are subsequently clinically suspected of
having the Richter syndrome (45).
Cytology. In CLL/SLL, a predominantly dispersed cell pattern
of homogenous population of small lymphocytes that are the
same size or slightly larger than a small, mature lymphocyte is
evident on smears. The nuclei are round and regular or slightly
irregular, with clumped chromatin, small to inconspicuous
nucleoli, and scant pale to slightly basophilic cytoplasm
(Fig. 12.12A and B). Occasionally, larger lymphocytes
representing prolymphocytes and paraimmunoblasts are
noted (Fig. 12.12C). These cells have centrally placed smooth
nuclei and abundant pale cytoplasm. The prolymphocytes
display single and central nucleoli while the paraimmunoblasts
contain either multiple, peripherally located nucleoli or a
single prominent nucleolus. Mitoses are rare and tingible body
macrophages or lymphohistiocytic aggregates are not seen.
FIGURE 12.12. Chronic lymphocytic leukemia/small lymphocytic lymphoma. A,B:
Homogenous population of small lymphocytes (slightly larger than RBCs) is evident on
smears (A, DQ stain, DS and B, Pap stain, DS ×60 objective). C: Nuclei are round and
slightly irregular with coarse clumped chromatin, small-to-­inconspicuous nucleoli, and
scant basophilic cytoplasm (C, DQ stain DS ×100 objective with oil immersion).
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Tingible (stainable) bodies are the phagocytosed apoptotic
cells and cell debris in the macrophage cytoplasm. In cases of
Richter syndrome, there are large numbers of blastic cells with
round nuclei with vesicular chromatin and prominent nucleoli
and scant basophilic cytoplasm (2,15).
Phenotype. Positive for CD5, CD23, and Pax5 (Fig. 12.10B)
and negative for CD10, FMC-7, and CD79b. CD20 and surface
immunoglobulin (sIg), light-chain expression are typically dim.
The proliferation index is usually low (<10%) with proliferation
marker Ki-67. A proliferation index of >30% may indicate
transformation (42). A newer marker LEF-1 is positive in
approximately 100% of CLL/SLL (46). A subset of DLBCL and
grade 3 FL are also positive for LEF-1 (46).
Cytogenetics. Deletion 13q14 (50%), deletion 11q (20%), and
trisomy 12 (10% to 15%). 17p deletion is associated with
disease progression and increased risk of death and is treated
differently (47).
Differential Diagnosis. Reactive
lymphadenitis and other
mature SLLs and large cell lymphomas when there is Richter
transformation.
Lymphoplasmacytic Lymphoma
Key Cytologic Features
• Small lymphocytes
• Plasmacytoid lymphocytes
• Mature PCs with Dutcher and Russell bodies
• Mast cells
• Epithelioid histiocytes
Clinical Features. Lymphoplasmacytic lymphoma (LPL) is an
uncommon low-grade B-cell malignancy comprising <2% of all
NHL. The disease affects older men. LPL usually presents with
bone marrow involvement, splenomegaly, and generalized
lymphadenopathy. The clinical course is generally indolent, but
transformation to large cell lymphoma occurs in 5% to 10% of
cases (48).
Cytology. Smears depict a mixed population of small lymphocytes
(similar to CLL/SLL) and plasmacytoid lymphocytes. The latter
have eccentric nuclei with coarse chromatin and inconspicuous
nucleoli. The chromatin, however, is lymphocyte-like and does not
demonstrate the “cartwheel” pattern as seen in PCs. Cytoplasm
is moderate and basophilic with occasional paranuclear “hof”
(large Golgi zone) (Fig. 12.13). Mature PCs containing Russell
bodies (cytoplasmic globular eosinophilic immunoglobulin
inclusions) and Dutcher bodies (intranuclear immunoglobulin
inclusions) are also seen. Occasional multinucleated cells may be
present. Mitoses are rare. Increased mast cells and epithelioid
histiocytes may be seen (15,49,50).
Phenotype. Positive for CD20, Ig light chains, and IgM and usually
positive for BCL2 and negative for CD5 (usually), CD10, CD23,
Cyclin D1, and BCL6. Lymphoplasmacytoid cells are positive for
CD38, and mature PCs are positive for CD38 and CD138.
Cytogenetics. Lacks a distinct molecular genetic hallmark.
Deletion of 6q21-q23, a nonspecific finding, is the most common
aberration reported in 40% to 70% of patients (50).
Differential Diagnosis. Distinguishing the LPL from other
small cell lymphomas, especially marginal zone lymphoma
(MZL) with plasmacytic differentiation, may be problematic.
PC neoplasms are also in the differential (50).
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FIGURE 12.13. Lymphoplasmacytic lymphoma. Small lymphocytes (similar to CLL/SLL) and
plasmacytoid lymphocytes. The latter has eccentric nuclei, lymphocyte-like coarse chromatin, and inconspicuous nucleoli. Cytoplasm shows occasional paranulear “hof” (DQ stain,
DS ×60 objective).
Plasmacytoma
Key Cytologic Features
• Mature and immature PCs
• Eccentric nuclei, “cartwheel” chromatin
• Basophilic cytoplasm, perinuclear “hof”
• Some binucleated cells
• Dutcher bodies and Russell bodies
• Mitosis
• Amyloid
• Blastic morphology in plasmablastic form
• Anaplastic morphology in anaplastic form
Clinical Features. PC neoplasms encompass clonal PC pro­
liferations with a wide range of clinical manifestations
and behavior. In most cases, PC neoplasms are associated
with the production of a monoclonal immunoglobulin, or
M protein which is detectable in the serum and urine. PC
neoplasms constitute approximately 15% of all hematopoietic
neoplasms and are rare before the age of 40. Plasmacytoma
is a neoplasm of mature and immature PCs that presents as a
discrete tumor mass. They can be multiple osteolytic tumors
(multiple myeloma [MM]) or solitary. Solitary or extramedullary
plasmacytomas (EMPs) are rare, constituting approximately
3% to 5% of all plasma cell neoplasms (PCN) and have no
associated bone marrow or systemic disease. EMP can occur
in any organ; however, approximately 90% are found in the
head and neck region involving the upper aerodigestive tract.
EMP may also present as a metastatic deposit from another
soft tissue plasmacytoma or as a consequence of MM. Primary
plasmacytoma of lymph nodes is uncommon, and metastasis
should be excluded before making this diagnosis (51,52).
Cytology. Smears are dominated by PCs, with varying degrees
of maturity. These cells are arranged singly and in sheets
and clusters. The mature PCs show monomorphic round and
eccentric nuclei with condensed “cartwheel” chromatin and
indistinct nucleoli. Cytoplasm is abundant and basophilic
and shows a perinuclear “hof.” Binucleated cells are usually
present (Fig. 12.14A and B). The plasmablastic form has a
more blastic morphology with enlarged nuclei and bi- or
multinucleation, open chromatin structure, prominent nucleoli,
higher N:C ratio, and bare nuclei. Anaplastic plasmacytomas may
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FIGURE 12.14. Plasmacytoma. A,B: PCs with varying degrees of maturity. Mature PCs show condensed “cartwheel” chromatin. Occasional
binucleated forms are seen. Note the crisp nuclear features in the Pap-stained smear (A, DQ stain, DS and B, Pap stain, DS ×100 objective
with oil immersion).
show considerable pleomorphism and need to be differentiated
from DLBCL. Tumor cells may contain Dutcher bodies and
Russell bodies. Mitotic rate is high in the pleomorphic types.
Extracellular eosinophilic material representing amyloid may
be seen in some cases and will show apple-green birefringence
with Congo red stain on polarizing light microscopy (51,52).
Phenotype. PCs lose many of the surface markers of mature
B cells and are frequently negative for CD45 and CD20, but
usually express CD79a, CD38, and CD138. The tumor cells
synthesize Ig, most commonly of IgA and IgG isotype and show
light-chain restriction.
Cytogenetics. Translocations involving Ig genes. Many involve
the switch region of the immunoglobulin heavy chain gene,
consistent with the expression of IgA or IgG. The t(11;14)
(q13;q32) occurs in 25% to 30% of myelomas, leading to
overexpression of cyclin D1.
Differential Diagnosis. MZL and LPL, both of which may have
evidence of plasmacytic differentiation, should be considered
in the differential diagnosis of PCN. Ancillary tests usually help
with this differential. Reactive plasmacytosis is composed of a
polymorphous collection of PCs and other inflammatory cells.
Marginal Zone B-Cell Lymphoma
Key Cytologic Features
• Nodal MZL (N-MZL) and extranodal MZL of mucosa-­
associated lymphoid tissue (MALT lymphoma) are
­cytologically similar
• Heterogeneous lymphoid cells
• Monocytoid B cells
• Small-to-intermediate centrocyte-like cells
• Plasmacytoid cells (especially in MALT lymphoma)
• Occasional immunoblasts
• Few PCs
• Occasional tingible body macrophages and lymphohistiocytic aggregates
• Rare mitoses
Clinical Features. MZL is a low-grade indolent B-cell lymphoma
that is derived from monocytoid B cells. It is divided into two
types, extranodal MZL (MALT lymphoma) and N-MZL. Both
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types are morphologically and immunophenotypically similar.
N-MZL is rare and has no extranodal or splenic disease.
MALT lymphoma constitutes 7% to 8% of all B-cell lymphomas and is more common in older women, but may also
occur in children. Up to half involve the gastrointestinal tract,
with stomach being the most frequent site. However, other
sites, not usually thought of as mucosa associated, can also be
involved including salivary glands, thyroid, lung, breast, and
urinary bladder. Most MALT lymphomas are associated with
a predisposing infectious or autoimmune condition, such as
Helicobacter pylori–associated chronic gastritis, Hashimoto
thyroiditis, and Sjögren Syndrome (50).
Cytology. Smears show a dispersed cell pattern comprising of
various proportions of lymphocytes with monocytoid B cells and
small-to-intermediate cells with centrocyte-like morphology.
The monocytoid B cells are medium-sized lymphoid cells with
round, reniform, or indented nuclei, with condensed chromatin
and small indistinct nucleoli. Cytoplasm may be scant to
abundant, and pale staining or clear. Small- to intermediatesized lymphoid cells contain slightly irregular nuclei and small
nucleoli and are thus centrocyte-like (Fig. 12.15A and B).
Plasmacytoid cells, scattered immunoblasts, and monoclonal
PCs are also seen. The latter are most common in the MALT
type. Lymphohistiocytic aggregates are common, while tingible
body macrophages may be seen in some cases. Mitoses are
rare. MZLs can transform to DLBCL in about 10% of the
cases (53–55).
Phenotype. Positive for CD19, CD20, CD79a (Fig. 12.15C), and
bcl-2 and negative for CD5, CD10, CD23, BCL6, and cyclin D1.
A subset is also positive for IgD (53).
Cytogenetics. t(11;18) is seen in up to one third of low-grade
extranodal MZL and trisomy 3 has been described in both
nodal and extranodal MZL (50).
Differential Diagnosis. Reactive lymphocyte proliferation and
other small B-cell lymphomas should be considered in the
differential diagnosis. Cytomorphology, specific phenotypic,
and genetic features, such as absence of coexpression of
CD19/CD5, seen in SLL/CLL and MCL, cyclin D1 expression in
MCL, CD10 expression in FL, and the characteristic t(14;18)
translocation involving the BCL2 locus in FL and the t(11:14)
translocation in MCL may be helpful in this regard (53).
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FIGURE 12.15. Nodal marginal zone lymphoma. A,B: Dispersed cell pattern comprising
various proportions of lymphocytes with monocytoid B cells and small-to-intermediate cells
with centrocyte-like morphology (A, DQ stain, DS ×100 objective with oil immersion and
B, Pap stain, DS ×60 objective); C: Positive immunostain for CD20 (DS ×60 objective).
Follicular Lymphoma
Key Cytologic Features
• Polymorphous lymphoid cells with centrocytes and
­centroblasts
• Lymphoid cell aggregates in one third of cases
• Centrocytes (cleaved cells) predominate
• Approximately 10% centroblasts (large transformed
­noncleaved cells)
• Lymphoglandular bodies (cytoplasmic fragments of
­lymphocytes)
• Reactive T lymphocytes
• Increasing number of centroblasts with increasing grade
• Increased mitoses with increasing grade
Clinical Features. FL is a B-cell neoplasm of follicular center cell
origin, comprising of two cell types found in normal germinal
centers, centrocytes (cleaved cells), and centroblasts (large
transformed noncleaved cells). It is a common form of NHL in
adults and accounts for about 20% to 40% of cases. Median age
at presentation is 55 years with equal sex incidence. The vast
majority of cases present with advanced disseminated disease
with spleen, bone marrow, and lymph node involvement
(33,56,57). Transformation to an aggressive large B-cell lym­
phoma may occur in 25% to 30% and is associated with rapid
progression (34,58).
The WHO recommends a three-tier grading system (grades
1 to 3) for FLs based on the absolute number of centroblasts
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per high-power microscopic field (hpf) in 10 neoplastic follicles
on histology (34). Grades 1 and 2 FLs, often combined together,
are indolent tumors and may not be treated, whereas grade 3
FLs are aggressive but potentially curable with chemotherapy.
Since FL may also have a diffuse growth pattern both The WHO
2008 and REAL classifications also recommend that the degree
of follicle formation (predominantly follicular, follicular, diffuse,
and predominantly diffuse) should also be reported. Grade 1
has an average of <6, grade 2, 6 to 15, and grade 3 >15 centroblasts/hpf counted in 10 neoplastic follicles. This method of
grading system has prognostic implications (34).
Cytology. On FNA smears, a loose follicular pattern or dense
lymphoid aggregates suggest follicle formation in these
lymphomas (Fig. 12.16A). Lymphoid cells are polymorphous
and consist of two cell types, the centrocytes and centroblasts
(Fig. 12.16B). The predominant cells are centrocytes. These are
small- to intermediate-sized lymphocytes (slightly larger than
benign lymphocytes) with cleaved nuclei with deep folds in
the membrane, occasionally imparting a bilobed appearance.
Chromatin is coarsely clumped and nucleoli are inconspicuous.
Cytoplasm is scant and pale or lightly basophilic with poorly
defined borders (Fig. 12.16C and D). The centroblasts, constituting
about 10% of the cells, are large noncleaved lymphocytes (twice
the size of benign lymphocytes), with round-to-oval nuclei, with
occasional indentation. Chromatin is reticular and nucleoli may be
single and central or multiple and peripheral. Cytoplasm is scant
and dense basophilic or amphophilic. The nuclear membrane
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms305
FIGURE 12.16. Follicular lymphoma. A: Loose clusters of small lymphoid cells indicating follicle formation (DQ stain, DS ×40 objective).
B: Lymphoid cells are polymorphous with predominant centrocytes with occasional centroblasts (B, DQ stain, DS ×100 objective with oil
immersion). C,D: Centrocytes are angulated or cleaved small lymphoid cells with deep nuclear folds (C, DQ stain. DS and D, Pap stain, TP ×100
objective with oil immersion).
abnormalities are similar to those identified in histologic sections
of small cleaved cell lymphoma, both follicular and diffuse.
Small mature reactive T lymphocytes are also seen. Numerous
lymphoglandular bodies (clumped cytoplasmic fragments) are
present. There are few or no mitoses in low-grade FL (56,57,59).
The relative lack of architecture in FNA remains a limitation in grading FL. Cytologic grading may be performed after
a morphologic and immunophenotypic diagnosis of FL (57).
Methods include counting centroblasts only in clusters resembling follicular structures and evaluating the proliferation
index by Ki-67 and DNA image analysis (60) or counting centroblasts, either in 300 lymphoid cells or per 10 hpf in TP slides
(61). Grading can also be performed on cell blocks, which may
better preserve follicular architecture (57). When the large
cells/hpf approach 20% to 50%, the cases may be classified
as grade II (mixed small cleaved and large cells) and when
the large cells constitute more than 50% of the cells/hpf, the
cases may be classified as grade III (variable mixture of large
noncleaved cells, large cleaved cells, and occasional immunoblasts) (2,57,58,60).
Phenotype. Positive for CD19, CD20, CD79a, CD10, and Bcl-6.
Approximately 85% are positive for Bcl-2. CD5, CD23, and
CD43 are negative.
Cytogenetics. t(14;18)(q32;q21) occurs in 95% of FL (62,63).
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Differential Diagnosis. RLH and MCL should be considered in
the differential diagnosis of FL grades 1 and 2. Low-grade
FL may be indistinguishable morphologically from normal
or reactive lymph nodes because of a mixed pattern of small
and large lymphocytes. This distinction between reactive
hyperplasia and low-grade FL poses a serious obstacle to
the use of FNA in the diagnosis of malignant lymphoma. FL
must also be differentiated from other small or mixed small
lymphomas including MCL. Morphologically, FL tends to be
less monotonous and contains a mixture of small and large
cleaved and large noncleaved cells, whereas MCL is composed
of predominantly small irregular cells. The tumor cells of MCL
are characteristically CD5 and cyclin D1 positive. FL-3 needs to
be distinguished from DLBCL (62).
Mantle Cell Lymphoma
Key Cytologic Features
• Monotonous small- to medium-sized lymphoid cells
• Irregular (centrocyte-like) nuclei
• Epithelioid histiocytes
• Lymphoid cell aggregates
• Rare plasmacytoid lymphocytes
• Rare tingible body macrophages
• Mitoses
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• Blastoid variant has intermediate to large cells—resemble
lymphoblasts
• Pleomorphic type has large cells—resemble centroblasts
Clinical Features. MCL is an aggressive B-cell lymphoma that
is uncommon, constituting approximately 5% to 10% of all
NHLs. These tumors mostly occur in older men. MCL is usually
widespread at presentation, with generalized lymphadenopathy,
hepatosplenomegaly, multiple lymphomatous polyposis within
the gastrointestinal tract, and bone marrow involvement.
Transformation to an aggressive blastoid variant is frequent
(33,34).
Cytology. Smears demonstrate a monotonous population of
small- to medium-sized lymphoid cells, slightly larger than
normal lymphocytes. Nuclei resemble centrocytes and are
oval and irregular containing dispersed mature chromatin and
inconspicuous nucleoli. Cytoplasm is scant and mildly basophilic
(Fig. 12.17A and B). The cell proliferation is homogeneous,
without the presence of larger cells with prominent nucleoli.
Epithelioid histiocytes with large oval nuclei and abundant
eosinophilic cytoplasm are commonly present. Lymphoid cell
aggregates are seen in one third of the cases. Plasmacytoid
lymphocytes and tingible body macrophages are rare. Mitoses
may be identified (53,64).
In the blastoid variant of MCL, the cells resemble lymphoblasts and are medium to large with irregular nuclei containing
finely granular chromatin and multiple irregular nucleoli. The
mitotic and apoptotic rate is high (Fig. 12.17C and D). Terminal
deoxynucleotidyl transferase (TdT) is negative (65). The pleomorphic type has large cells which resemble centroblasts.
Phenotype. Positive for pan B-cell antigens CD19, CD20, and
PAX5. MCL is also positive for CD79a, CD5, and FMC7 and
negative for CD10, BCL6, and CD23. The tumor cells express
monotypic sIg, with lambda light-chain restriction being more
common. Almost all show nuclear positivity for cyclin D1 that
provides a specific marker for MCL (66). SOX11, a transcription
factor for CNS development, is positive in >95% of MCL. It is
also found in Burkitt lymphoma (BL) and LBL and some grade
3 FL. SOX11 is not found in other small B-cell lymphomas (67).
Cytogenetics. MCL are characterized by t(11;14)(q13;q32)
resulting in BCL1 gene rearrangement with characteristic
overexpression of cyclin D1 in >95% of cases (47). In B-cell
NHL, cyclin D1 overexpression is limited to MCL and provides
a useful marker for this tumor (66).
Differential Diagnosis. Includes other small cell lymphomas
such as CLL/SLL, low-grade FL, and MZL. This differential
is important since MCL has a relatively worse prognosis.
Immunophenotyping may be critical in this differential. CLL/SLL
is CD23 positive and FL is CD10 and BCL6 positive. Difficulty in
diagnosing MCL can result from aberrant phenotypes. Positivity
FIGURE 12.17. Mantle cell lymphoma. A,B: Homogeneous population of small to medium-sized lymphoid cells with oval and irregular
nuclei, dispersed mature chromatin, inconspicuous nucleoli, and scant and mildly basophilic cytoplasm (A, DQ stain, DS and B, Pap stain,
DS ×60 objective). C,D: Blastoid variant with lymphoblast-like cells showing open coarsely granular chromatin (C, DQ stain, DS and D,
H&E-stained cell block ×100 objective with oil immersion).
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms307
for SOX11 may be helpful in the rare cases of CD5-negative/
equivocal or negative cyclin D1 MCL and CD5-positive MZL (67).
Diffuse Large B-Cell Lymphoma, Not Otherwise
Specified
Key Cytologic Features
• Monotonous population of large cells
• Several morphologic subtypes
• Centroblastic
• Immunoblastic
• T cell rich
• Anaplastic
• Epithelioid histiocytes and tingible body macrophages
• Necrosis, mitoses
• Artifacts of smearing
Clinical Features. DLBCL is characterized by a variety of
morphologic and clinical characteristics, suggesting an
underlying pathogenetic heterogeneity. DLBCL is a common
subtype of aggressive but potentially curable NHL that constitutes
approximately 30% of adult lymphomas. It can occur at any age
including childhood. Primary extranodal disease may be present.
Lymph nodes are also frequently involved. Most cases of DCLBL
arise de novo, while some cases represent transformation of a
pre-existing low-grade lymphoma. DLBCL may also occur in
patients with immunodeficiencies such as AIDS or may represent
a post-transplant lymphoproliferative disorder (59,68).
Cytology. DLBCL has a variable morphology. Smears of DLBCL
show a dispersed pattern of monotonous cells (Fig. 12.9A).
Usually, the smears are moderately cellular and recognizable as
abnormal due to the presence of large atypical. Morphology may
vary with the specific subtype—centroblastic, immunoblastic,
or anaplastic. The centroblastic type is most common. It
shows a preponderance of cells with medium-to-large cells
with round-to-oval nuclei with distinct smooth or irregular
contours, vesicular chromatin, and multiple prominent
nucleoli, often peripheral. Cytoplasm is scant basophilic
and may show vacuoles (Fig. 12.18A and B). There may be
admixed immunoblasts. The immunoblastic type is composed
predominantly of immunoblasts (>90%). Immunoblasts are
larger than centroblasts, have smooth round or oval nuclei,
and single central prominent nucleoli, a characteristic feature
of this subtype. Cytoplasm is abundant pale or clear to intensely
basophilic and may displace the nucleus eccentrically, imparting
a plasmacytoid appearance (Fig. 12.18C and E). Cells of this
type are seen in a large proportion of centroblastic lymphomas.
Anaplastic type shows large, occasionally multinucleated
pleomorphic cells (Fig. 12.18F and G). In the T-cell–rich type,
small mature T cells dominate the smears with a variable
FIGURE 12.18. Diffuse large B-cell lymphoma. A,B: Centroblastic type morphology with medium-to-large cells tumor cells, round-to-oval
nuclei with smooth or irregular contours, and basophilic cytoplasm with small vacuoles. Nucleoli are more obvious in the Pap-stained DS
(A, DQ stain, DS and B, Pap stain, DS ×100 objective with oil immersion).
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Knowles Neoplastic Hematopathology
FIGURE 12.18. (Continued) C–E: Immunoblastic type with larger cells, smooth round or oval
nuclei with single central nucleoli, and intensely basophilic cytoplasm. Note the nuclear features in the Pap-stained DS and TP (C, DQ stain DS; D, Pap stain, DS and E, Pap stain, TP ×100
objective with oil immersion). F,G: Anaplastic type showing pleomorphic cells (F, DQ stain,
DS ×60 objective and G, Pap stain, TP ×100 objective with oil immersion). H: Smearing may
induce cellular artifacts such as bare nuclei with many lymphoglandular bodies (cytoplasmic fragments) (DQ stain, DS ×60 objective). I: Poorly differentiated carcinoma may mimic
DLBCL. Note the vague syncytial arrangement of tumor cells, nonhmatolymphoid chromatin,
and absence of lymphoglandular bodies (DQ stain, DS ×60 objective).
admixture of histiocytes. Large tumor cells are scattered and
constitute <10% of all cells. All subtypes show variable numbers
of epithelioid histiocytes and tingible body macrophages. Cells
of DLBCL are fragile. Smearing may induce artifacts such as
loss of cytoplasm, which appears as lymphoglandular bodies,
and resultant distorted bare nuclei (Fig. 12.18H). Poorly
differentiated carcinoma (PDCa) cells may mimic DLBCL (Fig.
12.18I).
Phenotype. Cells of DLBCL express CD45 and B-cell markers
CD20 and CD79a. Most DLBCL also express surface or
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cytoplasmic Ig. The anaplastic variant expresses CD30.
DLBCL of centroblastic morphology frequently express CD10,
indicating a follicular center cell derivation. Expression for
BCL6 may also be seen. Large cell lymphomas have a falsenegative or nondiagnostic rate of approximately 27% by FCM
analyses. Possible causes of such a high rate include small
sample size, necrosis, or cell fragility (7,69,70).
Cytogenetics. Frequent molecular characteristics are 3q27
break involving BCL6 gene in 30%, BCL2 t(14;18) in 25%, and
MYC rearrangement in 10%. The latter may make it difficult to
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms309
distinguish DLBCL from Burkitt (34). Clonal rearrangement of
Ig heavy- and light-chain genes is also seen.
Differential Diagnosis. Tumor cells in the T-cell–rich subtype and
the anaplastic subtypes of DLBCL may resemble RS cells. Other
entities that should be considered in the differential diagnoses
include FL-3, blastoid variant of MCL, BL, and PDCa (Fig. 12.18I).
Immunophenotyping will aid in the differential (33,70,71).
Primary Mediastinal (Thymic) Large B-Cell Lymphoma
Key Cytologic Features
• Monotonous large neoplastic cells
• Enlarged irregular nuclei, granular chromatin, nucleoli
• Pale blue cytoplasm with distinct outer border
• High N:C
• Lymphoglandular bodies
• Mitoses
Clinical Features. Primary mediastinal large B-cell lymphoma
(PMBL), a unique subtype of DLBCL, is rare. It arises in the thymus,
the cell of origin is most likely the CD19+/CD21− thymic B cell
normally found clustering around Hassall corpuscles. The disease
usually affects young women and displays a unique combination
of clinical, histologic, immunologic, and cytogenetic features.
Patients commonly present with a rapidly enlarging mass in the
mediastinum that infiltrates surrounding structures and causes
compression symptom including superior vena cava syndrome. It
often involves the lungs and may eventually metastasize to other
organs. Bone marrow involvement is uncommon at diagnosis.
FNA is usually part of the initial workup of the patient.
Cytology. Smears show singly dispersed, highly atypical large-
to-intermediate, round-to-oval tumor cells. Nuclei are round
and eccentrically located with prominent nucleoli (plasmablastic
differentiation). The cytoplasm is scant to moderate in amount,
densely basophilic with occasional fine vacuoles and distinct
cell borders (Fig. 12.19A). Some cells are binucleated or
multinucleated (Fig. 12.19B). Plasmacytoid morphology is
more evident on air-dried DQ-stained slides (58,73,74).
Phenotype. Plasmablastic differentiation is confirmed by diffuse
and strong positivity for PC markers CD38, CD138, MUM1,
and PRDM1/Blimp-1. Lymphocytic and B-cell marker CD79a
is positive (50% to 85%), while others including CD45 and
CD20 and PAX5 are negative. EBER is positive and EBV LMP1
(Latency Pattern 1) is negative. The proliferation index with
Ki-67 immunochemistry approaches 100% (35).
Cytogenetics. Clonal Ig kappa light-chain gene rearrangement (73).
Differential Diagnosis. Plasmablastic myeloma must be distin­
guished from PBL because of the different clinical management
pathways. Typically, the presence of serum paraprotein and/
or lytic bone lesions in older patients favors MM. However,
widely disseminated bone disease and monoclonal serum Ig
have been described in PBL and could lead to major confusion
with MM. Although PBL and MM have nearly identical
Cytology. Smears show a monotonous population of atypical
lymphocytes, with predominance of large cells. The nuclei are
enlarged (>5 times the size of a normal lymphocyte), irregular,
cleaved or noncleaved, and lobulated, with coarsely granular
chromatin and occasional prominent nucleoli. Cytoplasm is pale
blue, scant to moderate in amount, and forms a narrow paleto-clear rim with a distinct cell border. Nuclear-to-cytoplasmic
ratio is high. Lymphoglandular bodies and mitoses are present
(72). FNA of primary DLBCL of the mediastinum may show low
cellularity due to sclerosis (69,72).
Phenotype. Distinct phenotype includes weak and nonhomogenous positivity for CD30 in 80% and CD23 positivity in
70% of cases. Expression of surface and cytoplasmic Ig is often
absent as is CD21 expression. BCL6 mutations and BCL2 gene
rearrangement are lacking.
Differential Diagnosis. Includes mediastinal involvement with
peripheral DLBCL, HL, anaplastic large cell lymphoma (ALCL),
thymoma, and carcinoma. PMBL can be distinguished from
these entities by ICC, FCM, and molecular features.
Plasmablastic Lymphoma
Key Cytologic Features
• Large tumor cells
• Round, eccentrically located nuclei, nucleoli
• Occasional bi- or multinucleated cells
• Basophilic or clear cytoplasm with distinct borders
Clinical Features. Plasmablastic lymphoma (PBL) is an uncommon
aggressive Epstein-Barr virus (EBV)–associated lymphoma of the
oral cavity, which is usually but not always associated with HIV.
Primary PBL may also occur at other extraoral sites, predominantly
in the gastrointestinal tract and lung (73). Rarely, PBL may also
occur in HIV-negative immunodeficient and immunocompetent
individuals. Such cases are more common in lymph nodes.
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FIGURE 12.19. Plasmablastic lymphoma. A,B: Singly dispersed atypical tumor cells with
plasmablastic differentiation; some cells are bi- or multinucleated (A, DQ stain, DS ×100
objective with oil immersion and B, DQ stain, DS ×60 objective).
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immunophenotypic profiles, CD56 positivity, occasionally cyclin
D1 positivity, and a relatively lower Ki-67 can help identify
secondary extramedullary plasmablastic MM (58).
Burkitt Lymphoma
Key Features
• Monomorphic medium-sized tumor cells
• Round nuclei, clumped chromatin, small nucleoli
• Deeply basophilic cytoplasm with small clear vacuoles
• Tingible body macrophages in a “starry sky” pattern
• Mitoses and apoptosis
Clinical Features. BL is an aggressive mature B-cell lymphoma
that may occur in extranodal sites. There are three major
subtypes of BL—endemic, sporadic, and immunodeficiency
associated. These subtypes of BL have similar morphology but
differ clinically and biologically. Endemic BL occurs in equatorial
Africa in areas of endemic malaria, strongly associated with the
EBV (95%) (75). It commonly occurs in children, 4 to 7 years old,
and affects mandible, gonads, and kidney. It is the most common
childhood malignancy in Africa. Sporadic BL is the most common
NHL in children and young adults in the developed countries.
It commonly presents as an intra-abdominal tumor. Lymphoid
tissue of Waldeyer ring may also be involved. Immunodeficiencyassociated BL is often associated with HIV infection and may
present with nodal and extranodal disease with bone marrow
involvement. BL is a fast-growing bulky disease. All types have
a high risk of CNS, ovary, breast, and kidney involvement. The
EBV genome is present in the neoplastic cells in all cases of
endemic BL. EBV is detected in approximately 33% of sporadic
and immunodeficiency-associated BL (75).
Cytology. Smears show a largely monomorphic population of
medium-to-large tumor cells. These cells bear round and uniform
nuclei with occasional clefts or indentations, clumped chromatin,
multiple small central nucleoli, and moderate amount of deeply
basophilic and vacuolated cytoplasm (Fig. 12.20A). Similar
cytoplasmic vacuoles can be seen in DLBCL (Fig. 12.18A).
Numerous tingible body macrophages impart a “starry sky”
pattern (Fig. 12.20B and C). Mitotic rate is brisk. Background
looks “dirty” due to individual cell necrosis (apoptosis) (76).
Some cases of BL may show tumor cells with plasmacytoid
features, particularly in immunodeficiency-associated BL, or
nuclear pleomorphism with more prominent nucleoli.
Phenotype. Tumor cells express light-chain restriction and
ger­minal center phenotype expressing CD10 and BCL6. Pro­
liferation index is high with nearly 100% positivity with Ki-67.
Cells are negative for CD5, CD23, TdT, and bcl-2.
Cytogenetics. t(8:14) in 90%. EBV genome can be demonstrated in
all endemic BL and approximately 30% to 40% of other subtypes.
Differential Diagnosis. Includes DLBCL and childhood small
round blue cell tumors. These can be distinguished by
immunostains and cytogenetic findings (77).
T-Cell and Natural Killer–Cell Lymphoma
T-cell lymphomas, which are more common in Asia, are a heterogeneous group of lymphomas with diverse morphology. In the
United States, they are much less common than B-cell lymphomas and constitute about 10% to 20% of all NHL. T-cell lymphomas are derived from post-thymic or mature T cells at different
FIGURE 12.20. Burkitt lymphoma. A: Monomorphic medium-to-large tumor cells with round
nuclei with occasional clefts and clumped chromatin. Note the small clear lipid-containing
cytoplasmic vacuoles (DQ stain, DS ×100 objective with oil immersion). B,C: Tingible body
macrophages impart a “starry sky” pattern (B, DQ stain DS ×100 objective with oil immersion
and C, Pap stain, TP ×60 objective).
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stages of differentiation and are divided into two broad categories in the WHO classification—precursor T-cell LBL and mature
T-cell and natural killer-cell lymphomas. T-cell lymphomas have
a poorer prognosis and response to treatment compared to
B-cell lymphomas. The section will cover only those entities that
may be encountered on FNA including precursor T-cell LBL/leukemia, PTCL (unspecified and angioimmunoblastic), and ALCL.
rearrangements. T-LBL almost always has T-cell receptor gene
rearrangements.
Precursor B- and T-Cell Lymphoblastic Lymphoma/
Leukemia (B- and T-LBL)
Peripheral T-Cell Lymphoma Unspecified (PTCL, NOS),
Including Angioimmunoblastic T-Cell Lymphoma
Key Cytologic Features in B- and T-LBL
Key Cytologic Features
• Small- or intermediate-sized blasts
• Round or highly convoluted nuclei, distinct nucleoli,
delicate blastlike chromatin
• Scant-to-moderate amount of nongranular cytoplasm
with frequent protrusions
• Tingible body macrophages
• Numerous mitoses
Clinical Features. Precursor T-cell LBL/leukemia are composed
of precursor B or T cells and involve the bone marrow and
peripheral blood occasionally accompanied by primary nodal
or extranodal site involvement. The neoplasm is rare. It
occurs in all age groups, but has a relatively high incidence
in children and young adults. B-LBL is most common in
children and may present with skin nodules, bone lesions,
and lymphadenopathy. T-LBL most frequently presents as
symptomatic mediastinal mass with pleural and pericardial
effusions and subdiaphragmatic lymphadenopathy. Both have
coexistent leukemic involvement (78).
Cytology. Smears contain small or intermediate-sized blasts with
round-to-convoluted nuclei with delicate blastlike chromatin,
distinct nucleoli, and scant-to-moderate weakly basophilic non­
granular cytoplasm (Fig. 12.21). Numerous mitoses are present.
Tingible body macrophages may be present (79).
Phenotype. Lymphoblasts in both B- and T-LBL are positive for
TdT. The T-LBL is positive for T-cell antigens CD2, CD3, CD5,
CD7, and CD1a and the B-LBL is positive for B-cell antigens
CD19, CD79a, and PAX5.
Cytogenetics. B-LBL show clonal but unmutated rearrange­
ments of Ig genes, two thirds show clonal Ig heavy-chain
FIGURE 12.21. Precursor T-cell LBL displays intermediate-sized blasts with nuclear irregularities (DQ stain, DS ×100 objective with oil immersion).
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Differential Diagnoses. Include BL and blastoid variant of MCL.
However, T-LBL is distinguished from B-cell lymphomas due to
absent B-cell markers and positivity for CD7 and other T-cell
markers.
• Small, intermediate and large lymphocytes
• Irregular nuclei, nucleoli
• Pale-staining cytoplasm
• Epithelioid histiocytes, PCs, and eosinophils
• Mitoses
• Reed-Sternberg (RS)–like cells
Clinical Features. PTCL is rare and constitutes <10% of all NHL
in the Western countries. It is primarily a disease of adults with
no sex predilection. Clinically, PTCL is a more aggressive form
of NHL. It is a heterogeneous group of tumors with variable
clinical presentation, morphologic appearance, and prognosis.
Patients commonly present with pruritus, lymphadenopathy,
and extranodal disease (80,81).
Cytology. PTCL most commonly shows a polymorphous pattern
with variable admixture of small, intermediate, and large
neoplastic cells. Percentage of large cells may range from 20%
to 90%. Smears in PTCL appear to be monomorphous when
one cell type predominates. The nuclei vary considerably
in size and exhibit significant membrane irregularities with
protrusions and indentations. Nucleoli may be present.
Cytoplasm is scant to moderate in amount, pale to basophilic,
and has well-defined borders (Fig. 12.22). Mitoses are
numerous. Epithelioid histiocytes, eosinophils, and PCs may be
seen. Occasionally, scattered large cells with vesicular nuclei
and prominent nucleoli, resembling RS-like cells, may be
observed (80,81).
Phenotype. Neoplastic cells express T-cell phenotype including
CD2, CD3, CD4, CD5, CD7, CD8, CD43 and CD45RO and are
FIGURE 12.22. PTCL shows large neoplastic cells with variably-sized nuclei with membrane
irregularities and scant cytoplasm (DQ stain, DS ×60 objective).
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negative for B-cell antigens. The RS-like cells may have similar
phenotype to RS-cells in HL with CD20, CD30 and occasionally
CD15 positivity.
Cytogenetics. Clonal rearrangement of T-cell receptor genes is seen.
Differential Diagnoses. Includes reactive hyperplasia, HL, ALCL
and DLBCL. A combination of cytomorphologic criteria, immun­
ophenotyping and molecular analyses are useful to distinguish
PTCL from these entities.
Anaplastic Large Cell Lymphoma
Key Cytologic Features
• Three variants: common, small cell and lymphohistiocytic
types
• “Hallmark” cells: large with irregular horseshoe-shaped
nuclei, prominent nucleoli,
• “Doughnut” cells: multiple nuclei arranged in a “wreathlike” pattern
• RS-like cells
• Histiocytes
• PCs
• Erythrophagocytosis
• Necrosis and inflammation
• Mitoses and apoptosis
Clinical Features. Ki-1 (CD30)-positive ALCL is a rare T-cell
lymphoma. ALCL can have diverse clinical, histologic and
cytologic presentation. It is divided into morphologically
indistinct but clinically distinct ALK-positive (ALK+) ALCL and
ALK-negative (ALK−) ALCL according to the presence or absence
of ALK protein. ALK+ ALCL is more common in children and
young men. It involves both lymph nodes and extranodal sites
(skin, bone, soft tissue, lungs, liver). ALK− ALCL tends to occur
in older patients with a lower male to female ratio and exhibits
immunophenotypic heterogeneity. Response to treatment and
prognosis of ALK+ ALCL patients is comparatively better than
in ALK− ALCL and other PTCL (82,83).
Cytology. ALCL shows a broad morphologic spectrum which
makes it difficult to make a definitive diagnosis on FNA (82).
The three distinct morphologic variants are the common large
cell and the less common lymphohistiocytic and small cell
patterns. The “hallmark” cells, so-called because they constitute
a hallmark of ALCL, are seen in all variants. These are large
pleomorphic cells with eccentric lobulated (horseshoe-shaped
or kidney-shaped) nuclei, finely granular chromatin, multiple
basophilic nucleoli and abundant basophilic or clear cytoplasm.
Usually a prominent perinuclear clear “hof,” corresponding to
the Golgi region is evident (Fig. 12.23A) (83). The other notable
cells are the “doughnut cells” which are multinucleated giant
cells, with nuclei arranged in a wreathlike pattern close to the
cytoplasmic border (Fig. 12.23B). RS-like cells are also present.
Background shows reactive lymphocytes, histiocytes, and PCs
(Fig. 12.23C). Erythrophagocytosis can occasionally be seen.
Necrosis, mitoses, and apoptosis are frequent. The small
cell variant shows medium-sized neoplastic cells with clear
FIGURE 12.23. Anaplastic large cell lymphoma. A: “Hallmark” cell with eccentric lobulated,
horseshoe-shaped, or kidney-shaped nucleus (Pap stain, DS ×100 objective with oil immersion). B: “Doughnut” cell with nuclei arranged in a wreathlike pattern (DQ stain, DS ×100
objective with oil immersion). C: Background shows reactive lymphocytes. Compare the size of
the neoplastic cells with the benign lymphoid cells (Pap stain, DS ×60 objective).
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cytoplasm and distinct cell membranes. Nuclei are irregular
with clumped chromatin resembling the horseshoe-shaped
nuclei of the classical “hallmark” cells. The lymphohistiocytic
variant contains similar small cells admixed with abundant
pale histiocytes (82).
Immunocytochemistry. Positive for ALK, CD30, EMA, CD45, and
one or more T-cell markers including CD2 and CD5.
Cytogenetics. T-cell receptor gene rearrangements,
translocation in >80% of ALK+ ALCL cases.
t(2;5)
Differential Diagnosis. Includes other large cell lymphomas,
anaplastic carcinoma, melanoma and HL. These tumors can be
distinguished based on morphology and ICC. Caution should
be exercised in interpreting positive EMA stain in a specimen
with carcinoma-like features as EMA is also a characteristic
marker for ALCL. ALCL and HL have overlapping morphologic
and ICC features. Careful evaluation of the latter two features
and clinical presentation would be helpful (84).
HODGKIN LYMPHOMA
WHO 2008 Classification of Hodgkin Lymphoma
• Classical HL
• Nodular sclerosis classical HL
• Mixed cellularity classical HL
• Lymphocyte-rich classical HL
• Lymphocyte-depleted classical HL
• Nodular lymphocytic predominant HL
HL is a relatively uncommon form of lymphoma (∼10% to
20%). The two distinct groups are the classical (CHL) and
the nodular lymphocytic predominant Hodgkin lymphoma
(NLPHL) types. These two groups differ in their epidemiology, clinical features, phenotype, genetics, and association
with EBV but share RS cells and its variants. CHL includes
four subtypes: nodular sclerosis (NSCHL), mixed cellularity
(MCCHL), lymphocyte rich (LRCHL) and lymphocyte depleted
(LDCHL) (85).
The role of FNA as a sole diagnostic modality in HL is controversial; however, FNA can be reliably used in the diagnosis
of recurrent HL. A cytologic diagnosis of HL is dependent on the
recognition of RS cells. Diagnostic RS cells may or may not be
easy to identify on FNA, depending on the histopathologic type
of HL. On FNA, the diagnostic accuracy of HL, not otherwise
specified, is >90%, but the accuracy of subtyping the disease is
lesser (86,87). However, accurate subtyping, as per WHO 2008,
is not considered crucial for management purposes. Rarely, RS
cells are seen in the absence of HL (88). FNA of HL may be
false-negative due to sampling error or fibrosis. When the findings on FNA do not correlate with the clinical and radiologic
impression, repeat FNA or histologic biopsy should be performed (89,90).
Key Cytologic Features
• Classical HL
• NSCHL may show low cellularity due to fibrosis
• Characteristic cells are classic RS cells or its variants
(Hodgkin cells)
• Background of inflammatory cells in NSCHL and
MCCHL
• Nodular lymphocytic predominant HL
• Characteristic cell are LP or “popcorn” cells
• Background shows small and large lymphocytes
• Histiocytes and PCs
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Classical HL
Clinical Features. Classical HL accounts for >95% of all HL and
shows a bimodal age distribution—younger and older age
groups with a male to female ratio of 2:1. Cervical lymph nodes
are the commonest site of presentation followed by mediastinal,
axillary, and inguinal lymph nodes. The spleen is involved in a
minority of cases. Other extranodal sites are uncommon except
in late stages. EBV infection is seen in 40% of CHL, with the
highest incidence in patients with AIDS.
NSCHL is the commonest subtype accounting for 70% of cases,
primarily affects young people, and shows a female predominance.
The mediastinum is involved in the majority of cases and more
than one half of patients present with stage II disease (86,87).
MCCHL constitutes about 20% of cases, while LRCHL and
LDCHL types are rare; the former constitute <5% of all CHL and
the latter <5% in the Western countries. MCCHL and LDCHL have
overlapping epidemiologic, clinical, and biologic features; both
are more common in developing countries, are most commonly
associated with HIV infection, and commonly involve peripheral
lymph nodes and bone marrow. MCCHL shows a bimodal age
distribution while LDCHL is more common in the elderly. These
three subtypes of CHL show a male ­predominance (85).
Cytology. The smears often show low cellularity in NSCHL due
to sclerosis. Small- to medium-sized lymphoid cells, eosinophils,
and other inflammatory cells with scattered, RS cells or their
mononuclear cell variants, the Hodgkin (H) cells, are present
(Fig. 12.24A and B). Classic RS cells are large, three to four times
the size of small lymphocytes and are binucleate or have a bilobed
nucleus with prominent eosinophilic nucleoli (often occupying
25% of the nuclear diameter), giving the cells an “owl-eyed”
appearance. Cytoplasm is moderate and basophilic (Fig. 12.24C–E).
The RS variant, Hodgkin cell, has a large nucleus, distinct
nucleolus, and abundant pale-blue cytoplasm (Fig. 12.24F). The
nuclei may be irregular and polylobated. If diagnostic RS cells are
identified in the appropriate polymorphous, dispersed population,
the diagnosis of HL may be rendered (Fig. 12.24A). In MCCHL,
H/RS cells are seen in a background of small- to medium-sized
lymphoid cells, some centroblasts, eosinophils, PCs, neutrophils,
and epithelioid histiocytes with occasionally granulomas. In
some cases of MCCHL, granulomatous inflammation may
dominate the smear and obscure the diagnostic cells. Smears
of LRCHL show H/RS cells in a background showing a spectrum
of lymphoid cells without eosinophils or histiocytes. In LDCHL,
H/RS cells dominate the smear (35).
Immunocytochemistry. CD15 and CD30 are positive (Fig. 12.24G
and H) and EMA, CD45, and usually CD20 are negative.
Positivity for EBV will be seen in EBV-associated cases.
Differential Diagnosis. Difficulties encountered in the diagnosis
of HL are attributable to overlapping features of RS cells and
a variety of benign and malignant cells. These RS cell mimics
or RS-like cells may be seen in large cell NHL (ALCL, T-cell–
rich B-cell lymphoma, PTCL), carcinoma (breast, gastric,
and nasopharyngeal), melanoma, sarcoma, germ cell tumor,
infectious mononucleosis (IM), and RLH (86,87).
ALCL and PTCL sometimes show large cells with large
nuclei and prominent nucleoli that resemble RS cells. Nuclei in
ALCL show prominent nuclear contour irregularities and cells
are negative for CD15. p63 protein may be used as a potential
tool in the differential diagnosis between ALCL and CHL. p63
is frequently expressed in a subset of ALCL cases while HL has
shown to be negative (89). Gene rearrangement studies can be
used to distinguish RS cells from T-cell lymphomas (90). The
occasional bizarre cells of nasopharyngeal carcinoma (NPC)
may mimic RS cells. Melanoma occasionally shows large, atypical cells that may mimic RS cells. MCCHL may be mistaken for
RLH if diagnostic RS cells are sparse or not noticed (12).
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A
B
C
D
E
F
G
H
I
J
FIGURE 12.24. Hodgkin lymphoma. Classical (CHL) in (A,B) shows a mixture of small and large lymphoid cells with scattered ReedSternberg (RS) cells (A, DQ stain, DS and B, Pap stain, DS ×60 objective). C,D: Diagnostic RS cells, also known as “owl-eyed” cell with
“mirror image” nuclei (C, DQ stain, DS and D, Pap stain, DS ×100 objective with oil immersion). E: Classic binucleated RS cell in an
inflammatory background (DQ stain, DS ×100 objective with oil immersion). F: Mononuclear variant of RS cell (Hodgkin cell). Note the
eosinophil and benign lymphoid cells (DQ stain, DS ×100 objective with oil immersion). G,H: Positive ICC for CD15 and CD30 (G and H, TP
×100 objective with oil immersion). NLPHL in (I,J) shows LP (popcorn) cell with multilobed nucleus and prominent nucleoli (I, DQ stain, DS
and J, Pap stain, DS ×100 objective with oil immersion).
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Nodular Lymphocytic Predominant HL
Clinical Features. NLPHL is rare accounting for <5% of all HL.
It differs from CHL in its clinical, epidemiologic, pathologic,
immunophenotype, and genetic features. It has a male
predominance and peaks in the fourth decade. The majority of
patients present with lymphadenopathy in the cervical, axillary,
or inguinal regions. NLPHL is rarely associated with EBV. It is
has a good prognosis even though recurrence is common (91).
Cytology. The LP cells are sparse and may be difficult to
find on FNA smears. The nuclei are folded or multilobated
with fine chromatin and distinct nucleoli (Fig. 12.24I and J).
Classical RS cells are not seen in NLPHL. The background
shows small and large lymphocytes. Histiocytes, singly or in
clusters, and PCs may be seen. Eosinophils and neutrophils
are not present (91).
Immunocytochemistry. The neoplastic cells of NLPHL are B cells
and express CD45, CD20, CD79a, and BCL6. They are also
positive for B-cell transcription factors PAX5, OCT2, and BOB-1
and negative for CD30 and CD15.
Genetics. Monoclonal rearrangements of the Ig genes.
Differential Diagnosis. Includes CHL and T-cell–rich B-cell
lymphoma (TCRBCL). CHL shows positivity for CD30 and
CD15. NLPHL is morphologically and immunophenotypically
similar to TCRBCL, and the differential diagnosis between the
two is controversial and a difficult task (97). Other entities
such as PTCL may need molecular studies to distinguish from
NLPHL (33).
HISTIOCYTIC AND DENDRITIC CELL
NEOPLASMS
Dendritic cells are antigen-presenting cells. Their main function is to process antigen material and present it on the surface
to other cells of the immune system. Dendritic cells include
Langerhans cells (on the skin), interdigitating dendritic cells
(IDDCs) (in the lymph node), and follicular dendritic cells
(FDCs) (in lymph node follicle). Neoplasms of dendritic cells are
rare. Langerhans cell histiocytosis (LCH) usually affects children between 1 and 15 years old and is traditionally divided
into three groups: unifocal, multifocal unisystem, and multifocal multisystem. The key to diagnosing LCH is the identification of the characteristic nuclear features of Langerhans cells,
FIGURE 12.25. A: Smear from a case of Langerhans histiocytosis showing large neoplastic cells with elongated grooved nuclei, prominent
nucleoli, and light blue cytoplasm. Note the eosinophils in the background (DQ stain, DS ×100 objective with oil immersion). Courtesy of
Stephan Papmbuccian, MD, Department of Pathology, University of Minnesota Medical Center, Minneapolis, MN. B: Dermatopathic lymphadenitis shows histiocytes containing melanin pigment, Langerhans cells, and scattered lymphocytes (Pap stain, DS ×60 objective).
C: Rosai-Dorfman disease shows a histiocyte with engulfed PCs (Pap stain, DS ×60 objective). Both (B,C) courtesy of Ricardo Bardales,
MD, Department of Pathology, Mercy General Hospital, Sacramento, CA. D: FDC sarcoma shows spiderweb-like network of thin, radiating,
multipolar processes interconnecting single tumor cells (Ultrafast stain, DS ×60 objective). Courtesy of Grace Yang, MD, Department of
Pathology & Laboratory Medicine, Weill Cornell Medical Center, New York, NY.
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which usually are admixed with numerous eosinophils. Langerhans cell nuclei have a folded, indented, or grooved appearance, generally with a fine chromatin pattern, inconspicuous
nucleoli, and a thin nuclear membrane (Fig. 12.25A). Dermatopathic lymph nodes may contain numerous Langerhans cells
and eosinophils; therefore, dermatopathic lymphadenitis is in
the cytologic differential diagnosis (Fig. 12.25B). FNA in sinus
histiocytosis with massive lymphadenopathy (Rosai-Dorfman
disease) shows large histiocyte-like cells with abundant pale,
eosinophilic cytoplasm containing well-preserved lymphocytes
and occasional PCs and granulocytes. The diagnosis can be
supported by the demonstration of S-100 protein in the large
cells (Fig. 12.25C) (92).
IDDCs sarcoma predominantly occurs in adults. Morphologically, IDDCs have a folded nucleus, in contrast to FDC sarcoma, which appear to have an elongated nucleus. On electron
micro­scopy, IDDCs have long and fine cytoplasmic extensions
that intermingle with similar cells. In contrast to FDCs, they
lack desmosomes, and in contrast to Langerhans cells they
lack Birbeck granules. Immunostains for S-100 and CD1a are
p
­ ositive.
FDC sarcoma also predominantly occurs in adults. On
FNA, the smears are cellular and show cells in syncytial fragments in addition to a prominent single-cell population. The
nuclei are round to oval and moderately pleomorphic with
vesicular chromatin, small nucleoli, occasional grooves, and
pseudoinclusions. Cytoplasm is abundant, eosinophilic, and
shows spiderweb-like network of thin, radiating, multipolar
processes interconnecting single tumor cells (Fig. 12.25D).
Delicate cytoplasmic strands are also seen in the small syncytial fragments, sprinkled with small lymphocytes. Positive
immunostaining for CD21, CD23, and CD35 confirms the
diagnosis (93).
Benign Conditions that Mimic Lymphoma
Reactive Lymphoid Hyperplasia
Key Cytologic Features
• Dispersed polymorphous lymphoid cells
• Predominantly small mature lymphocytes, centrocytes,
and centroblasts
• Immunoblasts
• PCs, eosinophils, neutrophils
• Tingible body macrophages,
• Lymphohistiocytic aggregates
• Mitoses
Clinical Features. Nonspecific RLH is usually of unknown cause
and frequently affects children and young adults. It is the most
common cause of lymphadenopathy in children and largely
attributed to the repeated antigenic stimulation encountered in
early life. In adults, RLH is less common. The enlarged lymph
nodes in RLH are usually < 3cm, soft, small, oval, mobile, and
single or multiple. RLH commonly involves the cervical, axillary,
or inguinal lymph nodes. The lymphadenopathy may last up
to a few weeks to months. In the clinical setting of persistent
or increasing lymphadenopathy, FNA may be warranted to
exclude lymphoma.
Cytology. FNA smears show a polymorphous population dom­
inated by small mature lymphocytes. The latter have round
nuclei and condensed dark chromatin. Other cells include
transformed lymphocytes including centrocytes, centroblasts,
and immunoblasts, scattered histiocytes including tingible body
macrophages, which are specialized histiocytes predominantly
found in germinal centers and PCs (Figs. 12.6A–C, 12.9B,
and 12.26). Lymphohistiocytic aggregates and mitoses are also
present (12,38).
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FIGURE 12.26. RHL shows a lymphohistiocytic aggregate comprising of small and large
lymphoid cells and tingible body macrophages. The latter are present both within and at
the periphery of the aggregate (Pap stain, DS ×60 objective). Figures 12.5A–C and 12.8B
depict other characteristics of a reactive lymph node.
Immunophenotype. Positive for pan-B-cell markers including
CD19, CD20, CD22, CD79a, and PAX5 with no light-chain
restriction nor any cytogenetic abnormalities, as well as panT-cell markers including CD3, CD2, and CD5.
Cytogenetics. Absence of t(14;18) is helpful in distinguishing
reactive from neoplastic proliferations.
Differential Diagnosis. In some lymphoid malignancies, a
heterogeneous lymphoid pattern may be seen. These include
low-grade FL, MZL, HL, and PTCL. These malignant entities
can be distinguished from RLH by FCM and molecular analyses
and by finding large atypical cells with CD15 and CD30
positivity in HL (12,38).
Granulomatous Processes
Some infectious processes may incite a granulomatous
response; however, clusters of epithelioid histiocytes, multinucleated giant cells, or a “sarcoid-like reaction” can be seen
in some malignant conditions affecting the lymph node. These
conditions include HL and NHL and metastatic tumors (94,95).
Histiocytes typically seen in granulomas have elongated
or curved nuclei with fine, evenly dispersed chromatin, small
nucleoli, and moderate amount of finely vacuolated cytoplasm.
Multinucleated histiocytic giant cells are commonly encountered (see specific types of granulomas in the Infectious Granulomatous Condition section below).
When granulomatous inflammation is diagnosed on FNA,
special stains for organisms, including acid-fast bacillus (AFB)
and Grocott methenamine silver stains, should be obtained.
Microbiology culture studies should be performed when ­possible.
Infectious Granulomatous Condition
Tuberculous Lymphadenitis
Key Cytologic Features
• Caseating granulomas
• Histiocytes, reactive lymphocytes, central caseous
necrosis
• Langhans giant cells
• Background of neutrophils and necrosis
• Intracellular and extracellular “negative” images of bacilli
on DQ stain in atypical mycobacteria (MAC) cases
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In TB, the most common finding, seen in about 50% of cases,
is the presence of epithelioid clusters with or without Langhans giant cells (large cells with nuclei arranged in a horseshoe-shaped pattern in the cell periphery) with necrosis
(Fig. 12.27A and B); necrosis is absent in about one third of
cases. FNA shows a sensitivity of about 75% in the detection
of tuberculous lymphadenitis (94). AFBs may be identified by
AFB special stain (Fig. 12.27C). In AIDS patient, MAC appear
as intracellular and extracellular “negative images” in air-dried
DQ-stained smears.
FIGURE 12.27. Granulomatous conditions. A–C: Necrotizing granulomatous lymphadenitis, secondary to tuberculosis: (A) A large, loose aggregate of epithelioid cells is
accompanied by a multinucleated giant cell and mixture of background lymphocytes
and neutrophils. (B) Note the necrotizing nature of the granuloma (A, Pap stain, DS ×40
objective and B, Pap stain, DS ×60 objective). (C) Positive special stain for acid-fast
bacilli (DS ×60 objective). D,E: Granulomatous lymphadenitis, secondary to sarcoid:
(D) Smear shows a loose aggregate of epithelioid histiocytes and (E) A multinucleated
giant cell with “asteroid bodies” (D, DQ stain DS and E, Pap stain, DS ×100 objective
with oil immersion).
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Noninfectious Granulomatous Condition
Sarcoidosis
Key Cytologic Features
• Noncaseating granulomas
• Epithelioid histiocytes, reactive lymphoid cells
• Multinucleated giant cells
• Cytoplasmic asteroid bodies or Schaumann bodies in
sarcoid
• No necrosis
Sarcoidosis is a systemic disease of unknown cause characterized by noncaseating granulomas. The disease predominantly affects adult and middle-aged African American
women. Although any organ can be affected, sarcoidosis
frequently involves mediastinal lymph nodes and lungs.
Cytologically, the main findings are well-formed noncaseating granulomas comprising of epithelioid histiocytes, multinucleated giant cells, and lymphocytes without necrosis
(Fig. 12.27D). Asteroid bodies (star-shaped intracytoplasmic
cytoskeletal elements) (Fig. 12.27E) and Schaumann bodies
(calcium oxalate crystals) may be observed. Sarcoidosis is
a diagnosis of exclusion; a foreign-body giant cell reaction,
an infectious etiology, or a malignancy need to be excluded.
Cytologic findings should be correlated with clinical and
imaging features (95).
Infectious Mononucleosis
Key Cytologic Features
• Polymorphous lymphocytes
• Immunoblasts
• Plasmacytoid lymphocytes
• Tingible body macrophages
• Occasional PCs
• Mitosis and apoptosis
IM is an acute self-limited disease caused by EBV. It is usually
seen in adolescents and young adults. In the acute infection,
the virus replicates in the perifollicular B cells, simulating a
vigorous humoral and cellular immune response. Typically,
patients present with fever, pharyngitis, and cervical lymphadenopathy (75).
Cytologically, IM displays large and small lymphocytes
and large numbers of immunoblastic cells than are usually
seen in a reactive lymph node. Immunoblasts are large cells
with a large nucleus and nucleolus and basophilic cytoplasm
(Fig. 12.28). Occasionally binucleated immunoblasts resembling RS cells are seen, a feature more commonly noted in
the tonsils. Plasmacytoid lymphocytes, tingible body macrophages, and PCs are also seen. Mitosis and apoptosis are
evident. In FNA smears, a polymorphic immunoblastic proliferation is suggestive of IM (Fig. 12.18A). ICC shows that these
are of both B- and T-lymphocyte phenotype. Serologic studies
confirm the diagnosis.
Primary and Metastatic Neoplasms
Primary and metastatic neoplasms including carcinomas, melanomas, germ cell tumors and sarcomas can mimic lymphoma
(38,96).
Poorly Differentiated Carcinoma
Key Cytologic Features
• Loosely cohesive cell clusters and single cells
• Cell crowding
• High-grade nuclei
• Less or no cytoplasmic differentiation
Metastatic carcinoma represents about 50% of cases in FNA
of lymph nodes. Well- to moderately differentiated carcinoma
usually shows cohesive cell clusters with community cell borders (Fig. 12.29A) and can easily be recognized as being of
epithelial origin on FNA. In adenocarcinomas, cytoplasmic
differentiation such as mucinous vacuoles may be present.
In squamous cell carcinomas, keratinized cytoplasm is usually seen. PDCa predominantly exhibit dispersed single cells
or loosely cohesive cell clusters and may mimic lymphoma.
Differential diagnosis of PDCa includes DLBCL (Fig. 12.29B).
A history of a previous carcinoma is usually present. Epithelial markers including cytokeratin in conjunction with
tumor-specific markers, such as thyroid-transcription factor
(TTF-1) for lung and thyroid, PSMA for prostatic, and CDX2
for gastrointestinal adenocarcinomas, can be applied to various types of cytology preparations. Artifactual aggregation
of cells in lymphoma may simulate carcinoma (Fig. 12.29C).
Small cell carcinoma may also be mistaken for lymphomas
(Fig. 12.30). Careful evaluation for the classic nuclear features of molding, “salt and pepper” chromatin with crush
artifact, and ICC positivity for endocrine markers including
chromogranin, synaptophysin, and CD56 help to distinguish
it from lymphoma (97).
FNA smears of metastatic NPC show neoplastic single cells
and clusters in a reactive lymphoid background (mixed pattern). Most single neoplastic cells present as large, pleomorphic
bare nuclei. Metastatic NPC and HL may have a similar reactive lymphoid background, with eosinophils, PCs, and sometimes epithelioid-cell granulomas, and can be distinguished by
ICC (98).
Melanoma
FIGURE 12.28. Infectious mononucleosis. Smear shows immunoblasts, reactive lymphocytes, and rare PCs (DQ stain DS ×100 objective with oil immersion).
Orazi9781609136826-ch12.indd 318
Melanoma demonstrates monomorphous mono- or binucleated cells in a dispersed cell pattern. Nuclei are large and
round with smooth chromatin and prominent nucleoli.
Cytoplasm is scanty and may contain melanin pigment (Fig.
12.31). The large binucleated melanoma cells with prominent nucleoli may mimic RS cells or cells of ALCL. Immunoreactivity for S-100 protein, HMB-45, and Melan A is
characteristic.
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Chapter 12 The Role of Fine Needle Aspiration Biopsy in the Diagnosis and Management of Hematopoietic Neoplasms319
FIGURE 12.29. Carcinoma. A: Well-differentiated carcinoma of lung depicting columnar epithelial cells in a glandular architecture (DQ stain, DS ×60 objective). B: Poorly
differentiated carcinoma may mimic DLBCL. In this case of poorly differentiated papillary thyroid carcinoma, cells are arranged in a vague follicular arrangement. Nuclei
are round to oval with occasional intranuclear inclusions and grooves. Cytoplasm is
pale and basophilic (Pap stain, DS ×60 objective). C: Cells of large cell lymphoma may
artifactually aggregate and may need ICC for confirmation of lymphoid nature (DQ
stain DS ×60 objective).
FIGURE 12.30. Metastatic small cell undifferentiated carcinoma of the lung. Aggregate of
cohesive tumor cells with nuclear molding, fine to coarse, evenly dispersed chromatin, and
scant to absent cytoplasm is easily identified. Note occasional nuclear elongation indicating
crush artifact and apoptotic bodies (DQ stain DS ×60).
Orazi9781609136826-ch12.indd 319
FIGURE 12.31. Metastatic melanoma showing dispersed neoplastic cells with eccentrically placed nuclei, prominent nucleoli, and granular blue-to-purple staining cytoplasm.
Bi- and multinucleation and intranuclear cytoplasmic inclusions were also noted (DQ stain
DS ×100 oil immersion).
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320
Knowles Neoplastic Hematopathology
FIGURE 12.32. FNA of seminoma shows the characteristic “tigroid background” and monomorphic tumor cells with hyperchromatic round-to-oval nuclei and prominent nucleoli and
lymphocytes (DQ stain DS ×100 oil immersion). Courtesy of Grace Yang, MD, Department of
Pathology & Laboratory Medicine, Weill Cornell Medical Center, New York, NY.
Small Round Blue Cell Tumors
A group of undifferentiated malignancies known as the small
round blue-cell tumors include neuroblastoma, Ewing family
of tumors consisting of Ewing sarcoma and primitive neuroectodermal tumors, rhabdomyosarcoma, and lymphoblastic leukemia. These tumors share cytomorphologic similarities that
can make them indistinguishable from each other and from
lymphomas.
On FNA, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma frequently form small cellular aggregates in addition to discohesive cells. Other specific architectural features may be evident
such as pseudorosettes in Ewing sarcoma and neuroblastoma
and neurophil-like material in neuroblastoma. Rhabdomyosarcoma nuclei tend to be round with a vesicular chromatin pattern,
occasionally with prominent nucleoli and relatively abundant
cytoplasm. Ewing sarcoma tends to have spherical to ellipsoid
nuclei with faintly stippled chromatin and small nucleoli.
ICC and cytogenetic studies can facilitate the distinction
between lymphoma and other types of small round blue-cell
tumors. Thus, for example, a panel composed of CD99 and
TdT, lymphocytic markers (LCA, CD20, CD3), and vimentin
appears to be sensitive and specific for distinguishing Ewing
sarcoma from LBL (99). Ewing sarcoma also shows cytogenetic
abnormality of t(11;22) in the majority of cases and t(21;22)
in a small number of cases. Immunoreactivity for CD56 and
chromogranin are effective in detecting neuroblastoma cells,
and desmin and myogenin are useful in rhabdomyosarcoma.
Seminoma
Dispersed monomorphic cells with thick nuclear membranes,
prominent nucleoli, and vacuolated cytoplasm with a lymphocytic component are characteristic for seminoma and may
simulate lymphoma. The “tigroid background” of seminoma,
produced by strands of cytoplasm and proteinaceous material,
provides a helpful clue on DQ stain (Fig. 12.32).
Immunoreactivity for nuclear OCT4, membranous c-kit, and
new marker SALL4 and 12p abnormality by FISH are seen in
seminoma (100).
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