Accuracy of diagnosis

The accuracy of FNAC of lymph nodes in the diagnosis of metastatic malignancy is influenced by many factors such as the size and site of the node, fibrosis, necrosis, previous irradiation and the number of punctures made. Small mobile nodes high up in the axilla are difficult to sample, while adequate material can easily be obtained from nodes only a few millimeters in diameter in a cervical or supraclavicular position.³⁷ Deep nodes are accessible to FNB by means of radiological imaging and guidance.^38,39 Fibrosis sometimes makes it difficult to obtain sufficient material for diagnosis from reactive inguinal nodes. It can also be a problem in nodular sclerosis Hodgkin lymphoma⁴⁰ and in some mediastinal and retroperitoneal non-Hodgkin lymphomas (NHL).⁹

Diagnostic sensitivity is occasionally limited by the fact that small metastatic deposits, metastases confined to the subcapsular sinus and single-cell metastases can be missed. However, early micrometastases rarely produce significant lymph node enlargement and if a lymph node is palpable it is likely to contain enough tumor tissue to be detectable by FNB. The diagnostic sensitivity of metastatic and recurring malignancy reported in the literature is usually above 95%.^23,41-53 Failure to obtain a representative sample is responsible for most false-negative diagnoses. Interpretation of a representative sample can be problematic, more often with lymphoma than with metastatic malignancy. For example, without immunophenotyping low-grade follicular lymphoma can easily be mistaken for reactive follicular hyperplasia.⁵⁴ Thus, although a negative cytological report makes malignancy unlikely, it is not singularly diagnostic,³⁷ and if the lymphadenopathy does not show signs of regression within a month or two, FNB should be repeated or a node should be excised for histology.

Diagnostic specificity for malignancy, on the other hand, is high. False-positive diagnoses are rare^{23,42,48,52-57} if particular caution is observed in the interpretation of smears from nodes in fields of previous irradiation and in the presence of necrosis. The existence of benign epithelial inclusions in lymph nodes (see p. 89) should be kept in mind. Again, the main problem is in relation to lymphoma, particularly the distinction between reactive follicular hyperplasia and follicular lymphoma. Most false-positive diagnoses are cases of reactive lymphadenopathy reported as suspicious of lymphoma.

Diagnostic accuracy not only depends on the aspirate being representative, but also very much on the quality of the cytological preparations. This is particularly the case in the diagnosis of some reactive lymphadenopathies and in the diagnosis and classification of lymphoma, which depends on the study of fine cytological detail in high magnification, and on an estimate of proportions of various cell types in the smear.^54,55,58-60 It is essential that the aspirates are handled and smears prepared by staff with experience in cytology to achieve satisfactory results.

There are conflicting opinions regarding the accuracy of cytological diagnosis and typing of malignant lymphoma.^{19-26,30,55,60-65} It is difficult to extract exact figures for several reasons. Most series include relatively small numbers of cases, with histologic correlation and/or consistent follow-up. Early series are based on cytomorphology (in FNB smears) alone, later series combine cytomorphology and immunophenotyping by flow cytometry and/or immunostaining of smears. The case mix (types of lymphoma; primary diagnosis or recurrent disease) is variable and often is not specified. With these reservations, it appears that the accuracy of diagnosing NHL by cytomorphology alone is in the range 60–80%, significantly lower for low-grade than for high-grade lymphomas.^{26,37,45,48,51,66}

Diagnostic sensitivity has generally been found to be lower for lymphoma than for metastatic malignancy.^23,48,67,68 For a diagnosis of lymphoma to be of clinically practical value, it must identify good and bad prognosis subgroups and therefore must specify the subtype. In an extensive review of the literature, two-thirds of the 30 studies reviewed, in which FNB was supplemented by immunophenotyping, diagnostic sensitivity was over 80% and specificity over 90%. However, only three of eight studies achieved a correct and precise subtyping in the primary diagnosis of NHL in more than 80% of cases.²² Difficulties in correlating cytology and histology of lymphoma have been enhanced by the parallel use and frequent modifications of different systems of classification over the years. The advent of the World Health Organization (WHO) lymphoma classification based on clinical, cytologic, immunophenotypic and genetic features has improved both the detection rate and classification of NHL, due to diminished importance placed on architectural features in subtyping NHL.^22,26,30,32 However, the accuracy of cytologic diagnosis is still limited in some forms of NHL, notably lymphomas with predominantly small cells, mainly marginal zone lymphoma, and in peripheral T-cell lymphoma, T-cell/histiocyte rich B-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma. This is also the case with the diagnosis of composite lymphoma and in the grading of follicular lymphoma.

The wide variation in the reported accuracy of diagnosis and typing of NHL probably reflects the environment in which the studies were carried out. A high accuracy has been achieved in a few highly specialized centers equipped with the full armamentarium and expertise related to ancillary laboratory techniques, but many hospitals have limited resources, and most pathologists still consider histology necessary in the primary diagnosis of lymphoma. This enables determination of any altered tissue and immunoarchitecture, and also reveals the cellular compartments and their composition in the tissue section, which is not reflected per se in the aspirated cell sample.

As a rule, FNB samples from malignant lymphoma are very cellular and can be used for ancillary studies.^{9,31,54,69-72} However, more material to allow a wider range of immune markers, assessment of tissue and immunoarchitecture as well as storage of material for ancillary studies and research can be obtained by supplementary CNB of the node, principally in intra-abdominal or intrathoracic nodal disease. With deep-seated masses, CNB is a useful adjunct to FNB. The FNB provides superior cytomorphology and is an excellent source for flow cytometry cell suspensions. This combined approach can increase diagnostic accuracy, assist in classification and reduce the number of insufficient samples.^73,74

The accuracy of cytological diagnosis of Hodgkin lymphoma (HL) is quite variable, frequently high, and in some studies greater than 85%.^22,23,75-77 The main problems in cytodiagnosis are in relation to the nodular lymphocyte predominant and lymphocyte-rich variants as well as separation from HL mimicks. The accuracy of subtyping HL in FNB smears is relatively poor.^22-24 Performing immunocytochemistry on cell block preparations, cytospins or NCB may help to a degree but a formal node biopsy is still necessary to provide reliable subtyping in most cases.

Complications

Significant complications do not occur. Post-aspiration hematoma or necrosis is rare.^78,79 To date, septic complications or tumor implantation in the needle track have not been reported following FNB of lymph nodes.

Technical considerations

Both reactive nodes and nodes involved by metastatic malignancy or lymphoma are highly cellular and moderately vascular tissues. Sufficient material is therefore easily obtained using 23–27-gauge needles, except sometimes in the presence of fibrosis or necrosis. FNB without aspiration has been used routinely for several years in some institutions. It has the advantage over the traditional aspiration of giving the operator a more direct and sensitive feeling of the consistency of tissues through the needle. This is helpful when small or deep nodes are biopsied. Non-aspiration also results in less admixture with blood. An abundance of blood adversely affects cell fixation and tends to cause shrinkage and distortion of cells. If aspiration is used, multiple rapid biopsies from different points of entry are preferable to multiple passes in different directions, in order to obtain a representative and adequate sample without too much blood.

Local anesthetic is not used and simple skin disinfection as for an injection is adequate. Two or more samples may be necessary to secure enough material for both routine smears and for special investigations and to reduce sampling error in focal disease. The use of gloves and extreme care in handling used needles are important safety precautions. The techniques involved in the biopsy of deep nodes using radiological guidance are described in Chapter 3. If the standard technique does not yield sufficient material, for example due to fibrosis (nodular sclerosis Hodgkin lymphoma and some sclerosing NHLs), a cutting core needle can be tried.^34,35 In general, large-caliber core needles of 14–18 gauge are recommended in the definitive diagnosis and typing of NHL, as they allow a more accurate assessment of tissue and immunoarchitecture as mentioned above. Artifactual change is less than with a 20-gauge needle.

It is not easy to make perfect direct smears from samples of lymphoid tissue and this takes considerable practice. An air-dried smear has to dry quickly for optimal fixation and has to be made thin and even. The smearing pressure must be well balanced to obtain a thin smear and at the same time avoiding crush artifacts. If the aspirate is bloody or thinned by a large amount of lymph fluid, cells need to be concentrated and separated as much as possible from the fluid. This can be achieved by using the two-step technique illustrated in Figure 2.7. A wet-fixed smear must be fixed immediately to minimize drying artifacts. Only those parts of the smears where the cells are evenly dispersed, well fixed and not distorted by the trauma of smearing should be chosen for diagnostic evaluation.

While air-dried MGG or Diff-Quik-stained smears are essential for the evaluation of cytoplasm and background elements, alcohol fixation and staining with H&E or Pap is helpful in assessing nucleoli and chromatin pattern. Whenever possible, both air-dried and wet-fixed smears should be made of each FNB sample as they may provide complementary information. Extra smears to allow cytochemical stains are often of great value. Staining for microorganisms (Ziehl-Neelsen, PAS, Gomori’s silver stain), mucin (PAS/diastase, Alcian blue), melanin (Masson-Fontana), and immunocytochemical studies are those most commonly used.

Heavy reliance is placed on ancillary techniques in FNB of nodes. A cell suspension made by gently dispersing the sample in Hank’s balanced salt solution with 10–20% fetal calf serum or by rinsing the needle with the fluid is a suitable preparation for this purpose. The suspension is spun in a cytocentrifuge at 300–700 rpm for 3–5 minutes. Processing should be done as soon as possible after biopsy since cell fragility increases rapidly with time. Cytospin preparations and cell blocks/cell buttons (see Chapter 2) allow assessment by a panel of immune markers by immunocytochemical staining as illustrated in the section on lymphoma. An elementary NHL screening panel could consist of CD20, CD3, CD5, kappa and lambda light chains, and this can be extended as appropriate. Background staining due to serum proteins and fragmented cells can be reduced by resuspending the cells in Hank’s fluid after initial centrifugation. Direct smears may be unsuitable due to the dispersal of cytoplasm and proteins in the background (see Chapter 2). However, immunostaining of FNB smear specimens can be utilized with the use of formal saline fixation. It eliminates background staining while allowing preservation of cellular antigenicity and morphology.⁸⁰ Immunocytochemistry is helpful in tracing the origin of metastatic malignancy, in the differentiation of lymphoma from reactive processes and from anaplastic carcinoma or melanoma, and in the classification of lymphoma.^13-1618 FCM analysis of aspirated cells is vital in immunophenotyping lymphoid populations, determining clonality and aberrant antigen expression. FNB material can be rinsed in a buffered balanced salt solution or RPMI solution to provide material for analysis. In addition, aspirated material can be used in conventional cytogenetics or fluorescence in situ hybridization (FISH) to assess for chromosomal translocations as well as in molecular studies/PCR to assess gene rearrangements.

If there is any suspicion of an infective process, the needle can be rinsed with sterile saline after the smears have been prepared, but preferably the biopsy should be repeated to provide sufficient material for culture for microorganisms (see p. 18 and Chapter 18).

Liquid-based preparations have been used minimally for nodal FNB samples. If used, they must be interpreted with caution as there are many cytomorphologic alterations.⁸¹

Indicators of the primary site

A stepwise approach to the investigation of nodal metastasis is suggested. This should include:

The patient’s age is an important consideration when assessing metastatic disease as certain tumors are more common in younger age groups, for example the small round blue cell group of tumors and nasopharyngeal carcinoma.

The anatomical site of the node may give some indication to the possible location of the primary tumor. For example, the axilla commonly harbors metastatic deposits from the breast, lungs or ovaries in middle-aged females. Nodes in the left supraclavicular fossa may be the site of presentation of pelvic malignancies, for example from the prostate, testis or ovaries as well as abdominal malignancies from the gastrointestinal tract.

The cytological patterns seen in routinely stained smears often give clues to the site of the primary tumor. Columnar cells with elongated nuclei arranged in palisades, stringy mucus and necrosis suggest a primary in the large bowel (see Fig 10.32), while mucin-containing signet ring cells suggest the stomach as the most likely primary site among several other possibilities. Glandular cells, moderately pleomorphic, arranged in a gland-in-gland or a cribriform pattern suggest prostatic carcinoma (Fig. 5.22). Large cells with abundant pale, granular or finely vacuolated cytoplasm and a low N : C ratio suggest a renal cell carcinoma. Very large central nucleoli are typical of less well-differentiated forms of this tumor and are also seen in large cell anaplastic carcinoma of lung and nasopharynx (see Fig. 5.55) and in hepatocellular carcinoma. Pulmonary and pancreatic adenocarcinoma can have a variety of patterns. They usually show a moderate degree of glandular differentiation, prominent nuclear pleomorphism and obvious mucin secretion. As a rule, the presence of intracytoplasmic mucin excludes renal, adrenal, hepatocellular and thyroid carcinoma. Breast cancer usually displays poor glandular differentiation while cell balls and single files of cells are more common. Nuclear pleomorphism is often relatively mild. Cells with intracytoplasmic neolumina containing ‘bull’s-eye’ inclusions are also suggestive of breast carcinoma.

Fig. 5.22 Prostatic carcinoma

Supraclavicular node aspirate containing malignant cells from a metastatic adenocarcinoma (MGG, HP).

The nuclei of a small cell anaplastic carcinoma of lung may appear large in air-dried smears but the amount of cytoplasm is minimal. Nucleoli are very small and indistinct in typical cases. The cells are closely packed together in aggregates or as single files with prominent nuclear molding. Pyknotic nuclei and nuclear debris are commonly seen between preserved cells (see Fig. 5.2), in the absence of massive necrosis. ‘Tear-drop’ nuclear artifacts caused by smearing are characteristic (see Figs 8.25 and 8.27). Merkel cell carcinoma (neuroendocrine carcinoma of skin) is characterized by mainly solitary undifferentiated small cells mimicking NHL. However, lymphoid globules are absent while CAM 5.2 and cytokeratin 20 staining is positive with a characteristic dot-like distribution (Fig. 5.23). Neuroendocrine markers (synaptophysin, chromogranin, CD56) are often positive. Features suggesting a neuroendocrine primary are: dispersal, ‘neuroendocrine anisonucleosis’, bland chromatin that may be speckled, cells in curved rows and poorly formed follicles (see Chapters 8 and 10).

Fig. 5.23 Merkel cell carcinoma

(A) FNB lymph node with predominantly dissociated undifferentiated tumor cells, superficially resembling NHL. However, lymphoid globules absent and lymphoid markers negative (MGG, HP). (B) Immunostaining of same case showing characteristic dot-like positivity of Merkel cell carcinoma with CAM 5.2 (HP).

Malignant melanoma is the great mimicker. Smears usually show total dissociation of cells, well-defined cytoplasm, eccentric nuclei, prominent anisokaryosis, a uniformly dense chromatin which does not vary much from nucleus to nucleus, often large nucleoli, binucleate cells, intranuclear vacuoles and often some cells with intracytoplasmic pigment (see Chapter 14). Malignant cells with abundant cytoplasm and a dark-staining paranuclear area are a clue in amelanotic melanoma. It can occasionally mimic lymphoma in smears (see Fig. 5.60), carcinoma when cellular groups are present, sarcomas with prominent numbers of spindle cells and even pleomorphic malignancies. Testicular tumors may be clinically occult and present with metastases to pelvic, para-aortic or supraclavicular nodes. The cytological pattern of seminoma is characteristic. The tumor cells are mainly dissociated and are mixed with lymphocytes and often with epithelioid cells. They have large, rounded, vesicular nuclei and an evenly distributed nuclear chromatin. Nucleoli are prominent but not very large in wet-fixed smears. The cytoplasm is pale and both cytoplasm and nuclei are very fragile, the dispersed cytoplasm forming a ‘tigroid’ background to the nuclei (Fig. 5.24, see also Fig. 5.27).^9,136 A tigroid background can, on occasions, also be seen in other glycogen-rich tumors: clear cell sarcoma, clear cell adenocarcinoma of the cervix and clear cell squamous carcinoma. Cells from a transitional cell carcinoma (TCC) may also be dispersed, resembling a large cell lymphoma (Fig. 5.25), or may form solid and sometimes papillary groups. The cells have abundant, relatively dense cytoplasm with distinct borders and pleomorphic, often eccentric, nuclei. A tendency to squamous differentiation or a spindle cell pattern (cercariform cells) may be seen, whereas papillary structures are uncommon in metastatic TCC.

Fig. 5.24 Lymph node metastasis of testicular seminoma

Smear from enlarged left supraclavicular lymph node. Mainly dispersed population of malignant cells with fragile nuclei, prominent nucleoli, and dispersed cytoplasm, intermingled with many lymphocytes (Pap, HP).

Fig. 5.25 Transitional carcinoma mimicking lymphoma

Dispersed malignant cells with large rounded nuclei, prominent nucleoli and scant blue cytoplasm. Irregular cytoplasmic fragments represent tumor necrosis, not lymphoid globules (MGG, HP).

Cytochemical stains and immune markers are often helpful. Squamous differentiation is most obvious in alcohol-fixed Pap-stained smears but can also be distinguished in MGG-stained preparations (Fig. 5.26A). Histochemical demonstration of intracytoplasmic mucin droplets confirms a diagnosis of adenocarcinoma. Positive immunocytochemical staining for prostatic acid phosphatase and/or prostate-specific antigen in an adenocarcinoma supports a prostatic origin.¹³⁷ This staining should be performed in all metastatic adenocarcinomas of unknown origin in male patients in view of the palliative treatment available for disseminated prostatic carcinoma.

Fig. 5.26 Squamous cell carcinoma

(A) Characteristic clear blue cytoplasmic staining, indicating squamous differentiation (MGG, HP). (B) Abundant dense cytoplasm and an occasional orangeophilic keratinised cell (Pap, HP).

Immunocytochemical staining for S-100, Melan A and HMB-45 is of great value in diagnosing melanoma. Immune markers with some ‘relative tumor specificity’ can be very helpful, e.g. PSA (prostate), TTF-1 (lung and thyroid), GCDFP-15 (breast and sweat gland, salivary gland) and PAX-2 (renal and mullerian). Other useful tumor markers are chromogranin, CD56 and synaptophysin in neuroendocrine tumors, a variety of hormones and polypeptides in endocrine tumors, glypican-3, Hep-Par1 and alpha-fetoprotein in hepatocellular carcinoma. Similarly, OCT4 and placental alkaline phosphatase (PLAP) are useful in some germ cell tumors, with CD117 in seminoma. Also, calcitonin and CEA in thyroid medullary carcinoma, and FLI-1 in primitive neuroectodermal tumor (see Table 2.4). CDX-2 is a colorectal carcinoma marker; however, it also reacts with some gastric and pancreatobiliary adenocarcinomas. Differential cytokeratins (CK7, CK20) may be helpful in suggesting the origin of metastatic carcinoma.^138,139 In some cases, electron microscopical examination of the aspirate and cytogenetic studies are extremely helpful, particularly in small round cell tumors and in some mesenchymal tumors (see Table 2.3).

Metastatic sarcomas are uncommon findings in lymph nodes.¹⁴⁰ Sarcomas other than Kaposi’s, which have a tendency to involve regional nodes, are rhabdomyosarcoma, epithelioid sarcoma, clear cell sarcoma, angiosarcoma, Ewing’s sarcoma and synovial sarcoma (Fig. 5.27)

Fig. 5.27 Nodal metastatic clear cell sarcoma

Tigroid background with round tumor nuclei and moderate cytoplasm.

Non-Hodgkin lymphoma^{9,10,11,38,41,54,55,58,60,68-70,84,141-145}

Today, most pathologists use the WHO classification of lymphomas (Table 5.1) which is based on a combination of cytomorphology, immunophenotype, genetic characteristics and clinical findings.⁶⁹ Lymphoma cell types, including Hodgkin lymphoma, are diagramatically illustrated in Figure 5.28. This classification allowed a greater role for FNAC with FCM in lymphoma diagnosis and subclassification. Cytological subtyping of NHL in FNB smears can be difficult and needs extensive experience and ancillary support. It requires a complete comprehension of the WHO classification of lymphomas (and/or hematopathology experience), a working knowledge of FCM and immunoperoxidase use and interpretation as well as cytogenetic and molecular studies in order to make the appropriate correlation with clinical information so that a classification can be made. There should also be close collaboration with hematopathologists/hematologists and senior people in the molecular, FCM and genetic laboratories.

Table 5.1 Entities of WHO classification of lymphoid tumours

Fig. 5.28 Diagrammatic representation of the most common cell types of malignant lymphomas.

FCM is the mainstay in the diagnosis and classification of NHL using FNB. FCM immunophenotyping provides quantitative evidence of cell lineage, B-cell clonality and T-cell aberrancies, and facilitates subclassification. FCM is not infallible and has pitfalls which may lead to misclassification or incorrect diagnosis. It is essential to correlate morphology with FCM data and the clinical situation.

The original 2001 WHO classification was revised in late 2008. It contains additions of new entities and subtypes (e.g. in the large cell lymphoma group), recognition of specific gray-zone areas and more degrees of acceptable uncertainty. More uncertainty has been recognized even within well-established entities, e.g. distinguishing lymphoplasmacytic lymphoma from other B-cell lymphomas with plasmacytic differentiation. These and other changes will add greater complexity to cytological subtyping of NHL. The number of possible diagnoses has more than doubled since the 2001 edition.

Over 90% of NHL are B-cell neoplasms, with diffuse large B-cell lymphoma and follicular lymphoma accounting for 65% of these. Less than 10% of NHL are T- and NK-cell neoplasms. As the B-cell lymphomas are commonly seen in daily practice, these will be highlighted, but not all of those listed in the WHO classification will be discussed. Some of the T-cell lymphomas also will be briefly discussed.

FNAC diagnosis of NHL is based largely on monomorphism of smear populations or disproportionate representation of cell types, often with features similar to their benign counterparts, which contrasts to most neoplasms diagnosed on the basis of cellular atypia. Assessment of cell size and nuclear features aid in subtyping. Small cells are less than 1.5 × the size of a normal lymphocyte. Medium sized cells are 1.5–2 × the size of a normal lymphocyte or no larger than a histiocyte nucleus. Large cells are more than 2–3 × the size of normal lymphocytes or larger than a histiocyte nucleus. The various cellular populations and cell size group entities are illustrated in Figure 5.29. The morphologic findings are then integrated with the immunophenotyping.

Fig. 5.29 FNA lymphoid populations: monotonous, heterogeneous and pleomorphic (Sm = small).

B-Cell neoplasms

Small cell lymphoid proliferations may have morphologic similarities and at times may be difficult to differentiate from reactive nodes. FCM is essential in distinguishing reactive from neoplastic small cell lymphoid populations as it demonstrates monoclonality in the neoplastic B cells, and aids in subtyping by assessing CD5, CD10, CD23 and FMC7 expression on the neoplastic B cells. This distinction can be difficult based on morphology alone (Table 5.2). Cytogenetics can also aid in subtyping.

Table 5.2 Small B-cell lymphomas

Small lymphocytic lymphoma/chronic lymphocytic leukaemia (SLL/CLL) (Figs 5.30-5.33)

Patients are mostly middle aged and elderly.

1 a monotonous population of small lymphoid cells,

2 mainly round nuclei slightly larger than those of normal small lymphocytes,

3 characteristically coarse granular nuclear chromatin (‘grumelé’, highlighted by Pap staining); nucleoli absent,

4 a varying number of prolymphocytes: larger size, more cytoplasm, pale chromatin, single central nucleolus,

5 large paraimmunoblasts with prominent nucleolus and gray-blue cytoplasm,

6 Rare tingible-body macrophages,

7 immunophenotype: CD19, CD20, faint SIg, CD5, CD23 (CD10 and FMC7 negative) (Fig. 5.30),

8 aberrant immunophenotype: CD5 or CD23 negative⁶⁹ (rarely CD 10 positive),

9 genetics: trisomy 12 (20%), deletion 13q (50%).

Fig. 5.30 Flow cytometric analysis of chronic lymphocytic leukemia

The upper left panel demonstrates CD5 and CD19 dual staining; note that quadrant 2 contains cells positive for both CD19 and CD5. Quadrant 1 contains the remaining CD5-positive T cells. The upper right panel demonstrates that the CLL cells are positive for CD23 (quadrant 4) and negative for CD10. The lower left panel demonstrates dual staining with CD19 and CD20. Note the typical weak CD20 staining on CLL cells. The lower right quadrant demonstrates the lambda light chain restriction, in that virtually all of the CD19-positive cells express (weak) lambda light chain.

(Courtesy A/Prof P. Macardle, Flinders Medical Center).

Fig. 5.31 Small lymphocytic lymphoma (B-CLL)

Monotonous population of slightly enlarged lymphocytes with coarsely granular chromatin (grumelé pattern) particularly obvious in Pap-stained smears; (A) MGG, HP; (B) Pap, HP.

Fig. 5.32 Small lymphocytic lymphoma (B-CLL)

Note the presence of many large cells with a single nucleolus (polymphocytes) representing proliferation center, especially in slightly understained cells (MGG, HP).

Fig. 5.33 Richter syndrome in B-CLL large B-cell lymphoma developing in terminal stage of B-CLL (MGG, HP).

Recognition of the subtle morphological differences between small lymphocytes and the small cells of lymphocytic lymphoma requires optimal cytological preparations. The typical SLL/CLL is readily recognized by the monotonous population of cells resembling small lymphocytes (Fig. 5.31). Difficulties can arise if the process contains numerous proliferation centers with many large and intermediate size cells – paraimmunoblasts and prolymphocytes (Fig. 5.32). A large B cell lymphoma may develop in 2–8% of patients with B-CLL, the so-called Richter syndrome (Fig. 5.33) or rarely HL.^9,69,145

Lymphoplasmacytic lymphoma (LPL) (Fig. 5.34)

Most patients are elderly and the majority of cases have a serum monoclonal IgM paraprotein.

Fig. 5.34 Lymphoplasmacytic lymphoma

(A) Predominantly small lymphocytes, some with plasmacytoid cytoplasm, and a single large blastic cell (MGG, HP) (Reproduced with permission from van Heerde et al.⁹); (B) Lymphoplasmacytic lymphoma with obvious plasma cell component (MGG, HP).

Criteria for diagnosis

1 a mixed population of lymphocytes, plasma cells and occasionally some blasts,

2 a variable number of lymphocytes with plasmacytoid features and Dutcher bodies,

3 may occasionally have amyloid,

4 immunophenotype: strong SIg and CIg, CD19, CD20, CD79a (CD5 and CD23 negative),

5 aberrant immunophenotype: occasionally CD5 and/or CD23 positive (rarely CD10),

6 genetics: no specific abnormality in node.

Occasional cases have a large number of immunoblasts. This may indicate a worse prognosis. Transformation to a large cell lymphoma is rare.⁶⁹ Other small cell lymphomas with plasmacytic features need to be excluded. At times marginal zone lymphoma may not be clearly separated from LPL.

Plasmacytoma (Figs 5.35, 5.36)^9,69,146,147

Usually in middle aged or elderly patients. Commonly involve bones, upper airways and only rarely lymph nodes.^9,71

Fig. 5.35 Plasmacytoma

Pure population of well-differentiated plasma cells (MGG, HP).

Fig. 5.36 Myeloma in lymph node

Poorly differentiated plasma cells. This patient had multiple myeloma (Pap, HP).

Criteria for diagnosis

1 cells resembling mature or immature (nucleoli-containing) plasma cells,

2 eccentric nuclei, condensed chromatin or blastic-like, condensed cytoplasm,

3 immunophenotype: CD38, CD79a, CD138, OCT-2, CIg; CD 20 and Pax 5 very often negative,

4 aberrant immunophenotype: CD56, CD10, CD117, cyclin D1.

Be wary of unusual cellular morphology, e.g. occasional cases dominated by pleomorphic, signet ring, clear and sarcomatoid cells, which may be difficult to recognize as plasma cells. The rare variant with a small lymphocyte-like morphology may mimic a B-cell lymphoma.

Follicular lymphoma (FL) (Figs 5.37-5.39)⁶⁹

Follicular lymphoma constitutes about 30% of NHL and is composed of a mixture of centrocytic and centroblastic cells. In the WHO classification, follicular lymphomas are divided into three histologic grades based on the number of large centroblasts present: grade 1 – predominantly centrocytes (0–5 centroblasts/HPF), grade 2 – a mixture of centrocytes and centroblasts, (6–15 centroblasts/HPF) and grade 3 – predominantly centroblasts (> 15 centroblasts/HPF).

Fig. 5.37 Follicular (grade 1) lymphoma

(A) Predominance of medium-sized cells with irregular, sometimes cleaved nuclei; a few centroblasts with multiple nucleoli. Relatively few small lymphocytes; (MGG, HP). (B,C) Kappa/lambda staining in cytospin preparation showing lambda monoclonality (B) (immunostain, alkaline phosphatase method, IP).

Fig. 5.38 Follicular (grade 1) lymphoma

Cytocentrifuge preparation; centrocytes showing cleaved nuclei; one centroblast (MGG, HP oil).

Fig. 5.39 Follicular (grade 1) lymphoma

Dendritic reticulum cells may be prominent in follicular lymphomas; predominance of centrocytes; (A, MGG, B, Pap, HP).

Criteria for diagnosis

1 lymphoid population composed of a mixture of centrocytes and centroblasts,

2 centroblasts with large round or slightly irregular nuclei, small nucleoli and basophilic cytoplasm,

3 centrocytes, small to medium-sized, with irregular or cleaved nuclei, inconspicuous nucleoli and little cytoplasm with a tendency to form clusters,

4 usually a relatively low number of small lymphocytes (Fig. 5.37A),

5 immunophenotype: monotypic SIg (Fig. 5.37B,C), CD19, CD20, CD10 (in 60–90% of cases), Bcl-2, Bcl-6, CD23 sometimes,

6 aberrant immunophenotype: CD5 (floral variant),

7 genetics: t(14;18) in over 90%.

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It is not possible in FNB smears to reliably predict the architectural pattern of a follicular lymphoma (FL). However, some authors have greater success in identifying aggregates of follicle center cells.¹⁴² A follicular pattern in cell blocks or NCB can be inferred by staining of dendritic cells with CD21/35. It is also difficult to assess the relative proportions of small and large centrocytes and centroblasts, as these are influenced by sampling bias. This makes grading difficult. Also, a clear distinction between FL grade 3B and DLBCL (mainly centroblastic) is often not possible. However, this distinction may not be critical for therapeutic management.

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There are no well-established criteria for grading FL in FNB smears. FL grade 3 has been variably defined,^21,29,145 for example, greater than 20%, or 50% centroblasts in the smear population, or there is sheeting of large cells. Grading has also been based on centroblast count with an associated proliferative index or proliferative index alone.^148,149 As a rule, this author favors FL grade 3 if half or more of the cell population, as assessed subjectively, are centroblasts.

In FL grade 1, the majority of cells are centrocytes. The characteristic cleaved nuclear morphology of centrocytes is well demonstrated in cytocentrifuge preparations of cell suspensions (Fig. 5.38). The irregular nuclear shape is often less obvious in direct smears and centrocytes are then identified by the intermediate size of the nuclei and by the nuclear chromatin, paler and more granular than that of lymphocytes. Starry-sky macrophages are usually absent. Dendritic reticulum cells are commonly seen (Fig. 5.39). FCM and/or immunohistochemistry are often necessary to distinguish follicular lymphoma from follicular hyperplasia in FNB smears. In 25–35% of FL there is transformation to a large cell lymphoma or high-grade Burkitt-resembling lymphoma. Rarely, they ‘transdifferentiate’ into various histiocytic and dendritic neoplasms.⁶⁹

Mantle cell lymphoma (MCL) (Fig. 5.40)^143,150,151

Usually in adults over 50 years of age

Criteria for diagnosis

1 monotonous population of small centrocyte-like cells with occasional histiocytes,

2 blastoid variant cells resemble lymphoblasts,

3 immunophenotype: CD19, CD20, SIg, CD5, FMC7, Cyclin-D1 (cell block or CNB); CD10 and CD23 negative,

4 aberrant immunophenotype: CD5 negative, CD5 and CD10 positive, CD23 weakly positive,^19,20,69

5 genetics: t(11;14) almost all cases.

Fig. 5.40 Mantle cell lymphoma

(A) Nuclei a little larger than those of lymphocytes irregular shape, finely granular chromatin without distinct nucleoli. A few normal lymphocytes present. (B) Central histiocyte for size comparison (C) Immunostaining with CD5: strongly positive reactive T cells and moderate positivity in malignant lymphoma cells (A, MGG, HP; B,Pap HP; C immunostain CD5, IP).

Mantle cell lymphomas are generally clinically aggressive neoplasms, usually high stage at the time of presentation: generalized lymphadenopathy, splenomegaly, bone marrow and blood involvement. One must be wary of some uncommon variants (pleomorphic, small round cell, marginal zone-like) which may overlap with other lymphoma subtypes.⁶⁹ The small cell variant appears to have a more indolent course. Immunostaining for Cyclin D1 on cytospin preparations can be unreliable.

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS)⁶⁹

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group. Those that do not belong to specific subtypes or disease entities are classified as diffuse large B-cell lymphoma, not otherwise specified (NOS). These make up 25–30% of NHL. There are three common variants: centroblastic, immunoblastic and anaplastic (pleomorphic). Not infrequently, the distinction between centroblastic and immunoblastic lymphoma is not clear, on morphologic grounds, due to a mixed population of large cells of different type.

Criteria for diagnosis

A Centroblastic variant (Figs 5.41-5.43)

1 a population of mainly centroblasts,

2 characteristically round nuclei with multiple small nucleoli (Fig. 5.41),

3 a variable proportion of indented/cleaved or even multilobated nuclei often present (Fig. 5.42-5.43),

4 immunoblasts with abundant basophilic cytoplasm and large central nucleoli may be present in the ‘polymorphous subtype’ (Fig. 5.43A).

B Immunoblastic variant (Figs 5.44-5.45)

1 a pleomorphic cell population dominated by large blasts; sometimes extreme pleomorphism and multinucleated cells,

2 unevenly distributed nuclear chromatin; prominent, usually single central nucleoli; occasionally multiple nucleoli; frequent mitoses,

3 abundant blue cytoplasm (MGG); perinuclear pale zone (Fig. 5.44A),

4 tingible body macrophages.

C Anaplastic variant

1 cells resemble Hodgkin and/or Reed-Sternberg cells,

2 may resemble cells of anaplastic large cell lymphoma.

Fig. 5.41 Diffuse large B-cell lymphoma, NOS

Predominance of large centroblastic lymphoid cells with pale nuclei, scanty cytoplasm and multiple, often peripheral, nucleoli (A, MGG, B, Pap, HP).

Fig. 5.42 Diffuse large B-cell lymphoma, NOS

Cytocentrifuge preparation of cerebrospinal fluid showing pronounced irregularity of nuclear contour (MGG, HP).

Fig. 5.43 Diffuse large B-cell lymphoma, NOS.

Polymorphous. Centroblasts, immunoblasts and large centrocytic cells, particularly distinct in Pap-stained preparations (A, MGG, HP; B, Pap, HP oil).

Fig. 5.44 Diffuse large cell lymphoma, NOS (immunoblastic B cells)

Predominantly large lymphoid cells with large round nuclei, large nucleoli and abundant basophilic cytoplasm (A, MGG, IP; B, MGG, HP oil).

(Reproduced with permission from van Heerde et al.⁹)

Fig. 5.45 Diffuse large cell lymphoma, NOS (immunoblastic B cells)

Predominance of large lymphoid cells with large vesicular round nuclei and large central nucleoli (Pap, HP).

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Diffuse large B-cell lymphoma, NOS, (groups A, B, C as above)

1 immunophenotype: CD19, CD20; often SIg, sometimes CIg; variable expression CD10, BCL6, BCL2 and MUM1; CD 30 most likely in anaplastic variant,

2 aberrant immunophenotype: CD5positve (10%); rarely cyclin D1 focal positivity,

3 genetics: t(14;18) in 30%, MYC rearrangement up to 10%,

T-cell/histiocyte-rich large B-cell lymphoma (Fig. 5.46)

Affects mainly middle-aged men.

Fig. 5.46 T-cell/histiocyte rich B-cell lymphoma

(A) Scattered large abnormal lymphoid cells with a background of reactive lymphocytes (MGG, HP); (B) The neoplastic B cell highlighted by immunostaining for CD20 (immunostain CD20, HP).

Criteria for diagnosis

1 smears dominated by small mature lymphocytes, over 90%,

2 variable numbers of epithelioid histiocytes,

3 scattered large cells mimicking Hodgkin or Reed-Sternberg cells, centroblasts, immunoblasts and ‘popcorn cells’,

4 immunophenotype: small cells, CD3, CD5, CD8; large cells, CD20, CD79a, BCL6 (CD15, CD30 negative),

5 genetics: non-specific.

It is difficult to diagnose on FNB smears and to distinguish from HL and reactive adenopathy^154-157 FCM is usually non-diagnostic. Usually a tissue biopsy with immune marker studies is essential to establish the diagnosis.

Primary mediastinal (thymic) large B-cell lymphoma (see chapter 9)

Plasmablastic lymphoma (Fig. 5.47)^69,158

Plasmablastic lymphoma is uncommon and may be associated with immunodeficiency.

Fig. 5.47 Plasmablastic lymphoma

(A) Uniform large cells with vesicular nucleus, prominent nucleoli and moderate cytoplasm-resembling immunoblasts (cell button. H&E, HP); (B) CD138 positive (immunostain CD138, HP).

Criteria for diagnosis

1 monotonous cellular smear with cells resembling immunoblasts or a mixture of large nuclei, coarse chromatin, small nucleoli and smaller plasmacytic cells,

2 immunophenotype: CD138, CD38, MUM1, CD79a (positive 50–85%),

3 genetics: frequent IG/MYC translocations and gains in multiple chromosomal loci.

It has an immunophenotype of plasma cells and needs to be distinguished from myeloma, principally on clinical and radiological findings.

Burkitt lymphoma (Figs 5.48-5.50)^159-161

Burkitt lymphoma in African children is endemic and associated with Epstein-Barr virus. The jaws often being involved. In non-African cases, most patients present with abdominal localization.

Fig. 5.48 Burkitt lymphoma

Rounded lymphoid cells; some variation in nuclear size; distinct nucleoli; granular chromatin; dense blue cytoplasm with lipid vacuoles and some starry-sky macrophages (MGG, HP).

(Reproduced with permission from van Heerde et al.⁹)

Fig. 5.49 Burkitt lymphoma

Blastic cells with round nuclei of varying size and small amount of cytoplasm; occasional starry-sky macrophages (Pap, HP).

Fig. 5.50 Burkitt lymphoma

Dual-color FISH with break-apart c-MYC probes ( 5’c-MYC probe orange and 3’c-MYC probe green) showing nuclei in which one pair of probe signals (yellow) is split apart due to a c-MYC region rearrangement.

(Courtesy Sarah Moore, Institute of Medical and Veterinary Science).

Criteria for diagnosis

1 usually a relatively uniform cell population with a high mitotic rate,

2 rounded nuclei of variable but predominantly intermediate size,

3 granular or speckled chromatin pattern, multiple (2–5) small but prominent nucleoli,

4 variable, mostly thin rim of dense blue cytoplasm with small lipid vacuoles (MGG),

5 starry-sky macrophages often prominent with a ‘dirty’ background,

6 immunophenotype: SIgM, CD19, CD20, CD10, BCL6 (negative for BCL2, TdT); proliferative index (MIB1) nearly 100%,

7 genetics: t(8;14) in 80%, t(2;8) and t(8;22) (Fig. 5.50).

The diagnosis of Burkitt lymphoma requires a combination of ancillary techniques as there is no single diagnostic gold standard, e.g. the c-myc rearrangement is highly characteristic but not specific.⁶⁹ In addition, there may be cases with greater nuclear pleomorphism with fewer and more prominent nucleoli.

B-lymphoblastic leukemia/ lymphoma (Fig. 5.51)^9,69,145,162

The majority of patients are under 18 years of age and the lymphoma often involves nodes, skin and bones. It comprises 10% of lymphoblastic lymphomas.

Fig. 5.51 B-lymphoblastic lymphoma

Rounded nuclei of variable, mainly intermediate size; speckled nuclear chromatin; small but distinct nucleoli. Starry sky macrophages may be present (A, MGG, HP; B, Pap, HP).

Criteria for diagnosis

1 a homogeneous population of cells of similar type.

2 round nuclei mainly of intermediate size, occasionally showing considerable variation in size,

3 finely granular or ‘speckled’ nuclear chromatin with multiple small nucleoli,

4 moderately basophilic, fragile cytoplasm,

5 starry-sky macrophages may be present (Fig. 5.51A),

6 immunophenotype: CD19, CD79a, CD10, PAX5, TdT (CD34, CD20 variable expression),

7 genetics: many with variable genetic abnormalities having prognostic significance.

This lymphoblastic morphology may be seen with T-cell lymphoblastic lymphomas

T-lymphoblastic leukemia/lymphoma (Figs 5.52, 5.53)

The lymphoma is frequently associated with a mediastinal mass and it comprises 90% of lymphoblastic lymphomas.

Fig. 5.52 T-lymphoblastic lymphoma

Note unipolar indentation (convolution) in some cells. Anisokaryosis, dense chromatin without distinct nucleoli, many mitoses. TdT positivity. (A, MGG, HP; B, immunostain,TdT, HP).

Fig. 5.53 T-lymphoblastic lymphoma

(A) Cerebrospinal fluid. In cytocentrifuge preparations the convolutions are very pronounced (MGG, HP oil); (B) Lymph node smear. Note mitotic figures (Pap, HP).

Criteria for diagnosis

1 a relatively uniform cell population with a high mitotic rate,

2 intermediate-sized nuclei, often prominent anisonucleosis,

3 variable number of convoluted (unipolar deeply indented) nuclei,

4 dense, finely granular chromatin; mostly inconspicuous nucleoli,

5 scanty, pale, fragile cytoplasm,

6 immunophenotype: TdT, most often CD3 (cytoplasmic) and CD7; often CD4 and CD8 double positive or double negative; variable positivity with CD1a, CD34, CD10,

7 genetics: 50–70% abnornal karyotype.

The convoluted, lobulated or segmented (cerebriform) shape is most striking in cytocentrifuge preparations of cell suspensions (Fig. 5.53A). It seems to be related to the mode of preparation to some extent and appears to be exaggerated by centrifugation. Lymphoblasts with convoluted nuclei often have markers for T cells but a similar morphology can be seen with some B-cell lymphoblastic lymphomas. Also, a similar nuclear complexity can be seen in several B-cell lymphomas after treatment. The cytomorphology alone is thus insufficient to classify the lymphoma as T-cell type and needs to be supported by immune marker typing.

Mature T and NK-Cell neoplasms

These lymphomas are more difficult to diagnose on FNAC than B-cell lymphomas as they do not have a specific immunophenotype, apart from anaplastic large cell lymphoma, ALK positive. In addition, FCM does not clearly demonstrate monoclonality as seen with B-cell lymphomas. However, immunophenotyping is essential to recognize T-cell lineage and aberrant T-cell phenotype, which is strong presumptive evidence of a monoclonal T-cell population. The loss of one or more of the pan-T-cell markers (CD2, CD3, CD5, CD7) is the most useful criterion in helping to make the diagnosis of T-cell neoplasia. Also suggestive are loss of both CD4 and CD8 or coexpression of both.^12,163 PCR for rearrangement of the T-cell receptor gene is often necessary to demonstrate clonality. Frequently tissue biopsy is necessary for initial diagnosis.

The cytology of peripheral T-cell lymphomas (PTCL) not otherwise specified (NOS) and NK/T lymphomas has been reported.^164-168 PTCL NOS have a varied morphologic appearance, commonly heterogeneous, with atypical medium to large lymphocytes (some Reed Sternberg-like cells (see Fig. 5.71) and an exuberant benign host inflammatory reaction, producing difficulty in its separation from reactive lymphadenopathy. Occasionally, there is a monomorphous proliferation of small, medium or large atypical cells. The nuclei are often indented, grooved or knobbly.

Angioimmunoblastic T-cell lymphoma (Fig. 5.54 and see Fig. 5.70A)^69,169,170

Typically seen with generalized lymphadenopathy, systemic symptoms and peripheral hypergammaglobulinaemia.

Fig. 5.54 Angioimmunoblastic lymphoma

Heterogeneous population of cells with small, medium and large lymphocytes, neutrophils, eosinophils and plasmacytoid cells (Diff-Quik, HP).

Criteria for diagnosis

1 heterogeneous cell population,

2 small to medium irregular lymphocytes,

3 variable number of large cells – including immunoblasts and at times pleomorphic cells (resembling Reed-Sternberg cells),

4 reactive background of small round lymphocytes, eosinophils, plasma cells, epithelioid and nonepithelioid histiocytes, dendritic cells,

5 immunophenotype: CD3, CD4, BCL6, CD10, CXCL13, PD1,

6 PCR: T-cell gene rearrangement detectable in 75–90% of cases; clonal immunoglobulin gene rearrangement in 25–30% of cases,

7 genetics: trisomy 3 or 5 most common.

Anaplastic large cell lymphoma (ALCL), ALK positive (Fig. 5.55)^{69,85,86,171-173}

Most frequent in childhood and young adults, presenting with lymphadenopathy.

Fig. 5.55 Anaplastic large cell lymphoma

(A), Large polymorphous cells with abundant cytoplasm; pseudoepithelial arrangement; multinucleation; distinct nucleoli. (B), Occasional ‘hallmark’ cells with horseshoe/boomerang nuclear forms. (C), CD30 (Ki-1) positive (A, MGG, IP; B, Diff-Quik, oil HP, C, immunostain CD30, IP).

Criteria for diagnosis

1 large cells with pleomorphic nuclei,

2 some Reed-Sternberg-like large cells,

3 irregular nuclei; horseshoe shaped (hallmark cells), donut, multinucleated with one or more prominent nucleoli,

4 cytoplasm abundant and may be vacuolated,

5 few lymphoglandular bodies,

6 cell clustering at times,

7 immunophenotype: CD30, ALK, EMA, one pan T-cell marker usually positive (null cell at times), perforin; variably positive for CD45 (CD15 and PAX5 negative),

8 genetics: t(2;5) in 84% of cases.

Several cytomorphologic variants exist, e.g. small cell, Hodgkin-like, sarcomatoid and lymphohistiocytic.⁶⁹ The ALK negative, anaplastic large cell lymphoma (ALCL) is seen in older adults and needs to be distinguished from HL (PAX 5 and cytotoxic markers being most helpful in making the distinction).

Problems and differential diagnosis (general)

1 suboptimal cytological preparations,

2 variable pattern in one node,

3 distinction from reactive lymphadenopathy,

4 NHL with few neoplastic cells in a dominant population of reactive lymphoid cells, e.g. T-cell/histiocyte rich B lymphoma,^155-157

5 small cell anaplastic carcinoma and other small cell tumors, particularly versus low-grade follicular (centrocyte dominant), lymphoblastic and Burkitt lymphomas,

6 large cell malignancies, undifferentiated carcinoma, melanoma, seminoma, myeloid sarcoma versus large cell lymphoma, including anaplastic large cell lymphoma,^{85,86,171-173}

7 limitations of ancillary techniques (PCR and FCM),

8 post-transplant lymphoproliferative disorders (PTLD),

9 gray zone (unclassifiable) lymphomas⁶⁹

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Direct smears must be made expertly, since poor preparation makes accurate diagnosis impossible. Suboptimal smears are the commonest cause of diagnostic difficulties and misinterpretations.

Sampling bias cannot always be avoided and may lead to an erroneous diagnosis. An aspirate from a node which is only partially involved with lymphoma may not be representative. Due to several mechanisms, the number of centroblasts and centrocytes does not always reflect the real composition within the lymph node. Thus, malignancy grading on cytological grounds is not reliable in follicular lymphomas.

The difference between normal lymphocytes and the neoplastic cells of small lymphocytic lymphoma is relatively subtle and the most obvious diagnostic feature is the monotony of the cell population. However, a sample including proliferation centers does not appear monotonous since there can be many cells of intermediate and large size (prolymphocytes and paraimmunoblasts^9,69 mixed with the typical cells of CLL type (see Figs 5.31 and 5.32). Such a case can be mistaken for reactive lymphadenopathy unless close attention is paid to fine cytological detail of the lymphocytes and to the result of immunocytology and FCM. Smears from small quiescent axillary and groin nodes may show a monotonous population of small lymphocytes. This can mimic small lymphocytic lymphoma, therefore this diagnosis should be avoided in small nodes unless there is immunologic proof of clonality.

Lymphoplasmacytic lymphoma (LPL) can also be mistaken for reactive lymphadenopathy in view of the sometimes heterogeneous character of the smear population. Again, a close study of cell detail usually reveals atypical lymphoid cells with plasmacytoid features. LPL is a diagnosis of exclusion, as other small cell lymphomas may have a mixture of relatively similar cells. At times, LPL cannot be separated from marginal zone lymphoma (MZL), as neither has a specific immunophenotype. MZL, with its mixed cell population, can mimic reactive hyperplasia. FCM, immunocytochemical, clinical and biochemical data are of utmost diagnostic importance.

In follicular lymphoma grade 1 there may be a high proportion of small lymphocytes to suggest a benign, reactive process. Also, the small centrocytes of some follicular lymphomas can be difficult to distinguish from lymphocytes in a reactive process, particularly if smears are not technically optimal. Nuclei must be studied carefully at high power to appreciate the slightly larger size, irregular shape and granular chromatin. Follicular lymphoma grade 1 with a predominance of small centrocytes is the type of lymphoma which is most likely to be missed cytologically and misinterpreted as reactive lymphadenopathy. FCM demonstrating B-cell clonality with CD10 positivity is important in making this distinction.

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At times, separating T-cell lymphomas with a polymorphous cell population from reactive hyperplasia can be difficult. In the evaluation, it is important to have the clinical history and presentation and to correlate it with the ancillary studies. Even then, a tissue biopsy is usually necessary to make a firm diagnosis.

In some cases of large cell lymphoma, the neoplastic cells may be relatively few in numbers, and the smears may be dominated by a background of reactive lymphocytes (Fig. 5.56). T-cell/histiocyte rich B-cell lymphoma is one important example (see Fig. 5.46).¹⁵⁵ Results of FCM may be difficult to interpret in such cases. Occasionally, distinction from reactive lymphadenopathy with scattered large, atypical-looking immunoblasts can be difficult.

Fig. 5.56 Diffuse large cell lymphoma, NOS, with reactive lymphocytes

Scattered neoplastic cells with very large nuclei with a dominant background population of reactive lymphocytes (MGG, HP).

In FNB smears from follicular lymphoma grade 1 and 2, the large centrocytic cells may have a tendency to clump together into aggregates showing some moulding of the nuclei. Rows and palisades of closely apposed, ovoid nuclei which appear columnar through moulding may simulate small cell anaplastic carcinoma or even adenocarcinoma (Fig. 5.57). However, the proportion of isolated cells is usually larger in lymphoma and many of these have the typical appearance of lymphoid cells with a rim of basophilic cytoplasm and a nuclear chromatin pattern which is different from that of carcinoma cells. Importantly, nuclei of lymphoma cells of a similar size to those of small cell anaplastic carcinoma usually have prominent nucleoli. This is a helpful but not infallible feature: nuclei of large centrocytes and of small cell carcinoma of intermediate type may be very similar in size and may have similar nucleoli. Nuclear molding and well-formed single files of tumor cells are more obvious in small cell carcinoma and other metastatic small cell tumors. The pale-blue, uniformly rounded cytoplasmic fragments characteristic of lymphoid tissue (lymphoid globules) are different from the cytoplasmic and nuclear fragments of tumor necrosis in smears of carcinomas (compare Figs 5.1 and 5.2). Immunocytochemical staining for cytokeratin, EMA and CD45 can usually solve the problem of distinguishing between small cell carcinoma and lymphoma in difficult cases. Additionally, FCM can help as small cell carcinomas are CD 56 positive and CD 45 negative.

Fig. 5.57 Pseudoglandular clusters in lymphoma

Such clusters may simulate small cell anaplastic carcinoma or adenocarcinoma; this problem mainly occurs in follicular lymphomas and large cell anaplastic lymphomas (A and B, MGG, HP).

Burkitt and lymphoblastic lymphomas occur commonly in childhood and need to be differentiated from small round blue cell tumors of childhood via morphologic, immunologic and cytogenetic means (see Chapter 17).

Smears of undifferentiated carcinoma (Figs 5.58 and 5.59, and see Fig. 5.25) and melanoma (Fig. 5.60) may show total dissociation of the tumor cells, nuclei may be large, with fine chromatin, prominent nucleoli and abundant basophilic cytoplasm. This pattern can be indistinguishable from DLBCL (particularly immunoblastic rich type) or ALCL, and at times even HL. Also, a dissociated seminoma may similarly be difficult to distinguish from a lymphoma. Myeloid sarcomas may be mistaken for DLBCL. They have dissociated large cells with pale chromatin, prominent nucleoli and rarely cytoplasmic granules. The presence of lymphoid globules in lymphoma and lobulated nuclei is helpful. Well-formed, sharply delineated aggregates of tumor cells, not just clumping of cells, are not seen in lymphoma. Again, in difficult cases, immunological staining for cytokeratin, Melan-A, S-100, OCT4, myeloperoxidase and lymphoid associated markers is usually decisive (Table 5.3).^{85,86,171-174}

Fig. 5.58 Small cell anaplastic (neuroendocrine) carcinoma

This example of a FNB smear from a lung tumor closely mimics anaplastic large cell lymphoma: dispersed cells with large eccentric nuclei and a thin rim of blue cytoplasm (MGG, HP).

Fig. 5.59 Nasopharyngeal carcinoma

Metastatic large cell undifferentiated carcinoma with solitary tumor cells may mimic large cell lymphoma (MGG, IP).

Fig. 5.60 Metastatic melanoma mimicking lymphoma

(A) Dispersed malignant cells with eccentric nuclei and pale cytoplasm; lymphoid globules absent (Pap, HP). (B) Amelanotic melanoma showing dense smooth chromatin and characteristic paranuclear dark area in tumor cells (MGG, HP).

Table 5.3 Large cell malignancies

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FCM results may be unreliable or noninformative if the aspirated material is necrotic or heavily admixed with blood. It must be remembered that B-cell clonality does not always equate with malignancy and its absence does not necessarily indicate a benign process. Polyclonality on flow cytometry may be seen in T-cell lymphoma, HL, partial involvement of a node by lymphoma, absence of immunoglobulin expression in some DLBCL NOS, occasionally marginal zone lymphoma as well as in a reactive lymph node.¹⁰ Close correlation of FNAC, immunologic and clinical findings is always essential. One must also be aware of aberrant immunophenotypes in B-cell NHL (see NHL overveiw) and also false-positive B-cell clones. An unusual, infrequent immunophenotype is the B-cell lymphoma coexpressing CD5 and CD10, such as follicular lymphoma, mantle cell lymphoma and DLBCL, NOS.¹⁷⁰ Rarely, with follicular hyperplasia in children and young adults there may be clonality.^175,176 The author has also seen similar false B-cell clonality in lymph nodes with metastatic melanoma and adenocarcinoma. Infrequently, in progressive transfomation of germinal centers, there may be coexpression of CD4 and CD8, causing confusion with a T-cell neoplasm.

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PCR false-negative results may occur with follicular, marginal zone and DLBCL NOS.^177,178 In contrast, small lymphocytic lymphoma and mantle-cell lymphoma have a much lower rate of false-negative clonal detection.^179,180 False-positive results may occur with some benign conditions, for example autoimmune diseases and some infectious processes.¹⁰

PTLD are more often extranodal in presentation, and there is a spectrum ranging from plasma cell hyperplasia/infectious mononucleosis-like reaction to diffuse lymphoma (predominantly B cell), however T/NK-cell and Hodgkin lymphomas also occur. FNB may provide a rapid diagnosis of monomorphic PTLD as it usually resembles B-cell neoplasms: DLBCL, Burkitt lymphoma and plasma cell neoplasms.^69,181-183 In polymorphic PTLD there is a mixture of small and large lymphocytes with plasma cells, which may resemble a lymphoma with plasmacytic differentiation or infectious mononucleosis. In this category, the diagnosis requires flow cytometry, PCR and usually histologic assessment.¹⁸¹

There are two main groups in the gray-zone (unclassifiable) lymphomas. Firstly, cases from a morphological and phenotypical perspective resemble Burkitt lymphoma; however, some feature(s) prevent the diagnosis. Secondly, cases may morphologically resemble DLBCL; however, the phenotype is that of HL or vice versa. These cases cause difficulty in subtyping with histologic specimens, and equally cause problems with cytologic samples.

Hodgkin lymphoma (HL) (Figs 5.61-5.68)^{40,75-77,184,185}

Hodgkin lymphoma accounts for approximately 30% of all lymphomas.⁶⁹ It is comprised of two entities: nodular lymphocyte predominant HL and classic HL. The subtypes of classic HL are nodular sclerosis, mixed cellularity, lymphocyte-rich and lymphocyte depleted. These account for up to 95% of cases of HL.

Fig. 5.61 Hodgkin lymphoma

Binuclear Reed-Sternberg cells and mononuclear Hodgkin cells in a background of mainly small lymphocytes (A) and/or granulocytes (B). Varying basophilia (A and B, MGG, IP).

Fig. 5.62 Hodgkin lymphoma

Many scattered binuclear or multilobated Reed-Sternberg cells with a background of lymphocytes (A and B Pap, HP).

Fig. 5.63 Hodgkin lymphoma

Characteristic CD30 positivity (immunostain CD30, IP).

Fig. 5.64 Hodgkin lymphoma

Examples of Reed-Sternberg cells from different cases; multiple or multilobated large nuclei with very large nucleoli; reticulate chromatin; pale cytoplasm (A and B, MGG, HP).

Fig. 5.65 Hodgkin lymphoma

Other examples of Reed-Sternberg giant cells (A, MGG; B, H&E, HP).

Fig. 5.66 Hodgkin lymphoma

Recurrent Hodgkin lymphoma; atypical mononuclear cell forms only (MGG, HP).

Fig. 5.67 Lymphocyte predominant Hodgkin lymphoma

Multinucleated ‘popcorn’ cell in a background of small B lymphocytes and some histiocytes (MGG, IP).

Fig. 5.68 Syncytial variant of Hodgkin lymphoma

Clusters of numerous large atypical lymphoid cells dominate smears focally; some are of Reed-Sternberg type (A and B, MGG, HP).

Classic HL, in nearly all cases, is derived from B cells. In nodular lymphocyte predominant HL, the Reed-Sternberg (‘popcorn’) cells are B cells reacting with CD20, OCT-2, BOB.1 and CD79a, and the small background lymphocytes are predominantly B cells but there are also many T cells. In classic HL the background lymphocytes are usually predominantly T cells, except in the nodular lymphocyte-rich variant where they are usually predominantly B cells. The Reed-Sternberg cells in these classic variants are often but not always negative for B-cell markers CD20 and CD79a. When positive for these markers, only a minority of these cells usually stains, and the staining intensity is varied. They are also weakly positive for PAX5.⁶⁹

A confident diagnosis of classic HL can only be made in the presence of typical Reed-Sternberg cells with a background of lymphocytes and reactive cells, as listed above, with its typical immunophenotype (Figs 5.61-5.65). Reed-Sternberg cells have large, lobulated nuclei which may appear symmetrically double (mirror nuclei) or complex and multiple. The nuclear chromatin is coarse and irregularly distributed in a reticular fashion with clear areas in between which give the nucleus an overall pale appearance. Nucleoli are large, often huge, eosinophilic in H&E preparations, pale grayish to dark magenta in MGG-stained smears. The cytoplasm is abundant, but pale and fragile so that the nucleus often appears to be surrounded by an empty space (see Fig. 5.64). Sometimes the characteristic nucleoli are not well demonstrated. Frequently only mononuclear cells which have a nuclear structure similar to the typical Reed-Sternberg cells (mononuclear Hodgkin cells, Fig. 5.66) along with their bare nuclear forms are present. In such cases, the definitive diagnosis must await histological examination, except in recurrent disease when classic Reed-Sternberg cells are not essential for diagnosis. The background cell population is mainly lymphocytes of reactive type, but polymorphs may be predominant in some cases (see Fig. 5.61B). The term ‘suppurative variant of HL’ has been proposed.¹⁸⁵

Histological examination is also necessary to distinguish confidently the five subtypes of HL. The two extremes, lymphocyte predominant and lymphocyte depleted, are, of course, reflected in the smears by the number of lymphocytes relative to the number of Reed-Sternberg and mononuclear Hodgkin cells. A suggestion of the presence of the lacunar type of Reed-Sternberg cell, characteristic of nodular sclerosis Hodgkin lymphoma in histological sections, is occasionally present in FNB smears. The tough consistency of the node felt with the needle, a scanty aspirate and the presence of fibroblasts and collagen fragments in the smear are features suggestive of the nodular sclerosis type. In the mixed cellularity type, typical Reed-Sternberg cells are usually easy to find and there are many plasma cells and eosinophils as well as histiocytes in the background cell population.¹⁸⁶

The nodular lymphocyte-predominant subtype is characterized by a monotonous population of slightly irregular small lymphocytes with relatively few scattered large pale multinucleated giant cells, corresponding to the ‘popcorn’ cells in histology, usually without distinct nucleoli (Fig. 5.67). In contrast to the other (classic) variants of HL, the bulk of the small cells and also the giant cells exhibit a B-cell phenotype.^9,69 This is a difficult diagnosis to make on FNB smears as there are usually few atypical cells in a sea of reactive small lymphoid cells and the architecture cannot be assessed.

Poor biopsy yield is a problem mainly in the nodular sclerosis subtype. Not infrequently, smears show only a few lymphocytes, fibroblasts and fragments of collagen, which may suggest a chronic inflammatory process. Multiple biopsies or the use of a cutting core needle may be necessary to obtain sufficient material.

Large, multilobated nuclei resembling Reed-Sternberg cells can be seen in a variety of conditions (Figs 5.69-5.74). Atypical immunoblasts in non-neoplastic reactive lymphadenopathy, for example in mononucleosis (Fig. 5.69), toxoplasmosis (Fig. 5.70B), rheumatoid arthritis-associated and drug-induced lymphadenopathy, may have nuclei of this type. They usually differ from the typical Reed-Sternberg cells by having smaller and darker nucleoli, a denser chromatin and a basophilic cytoplasm. Multinucleated giant cells in small or large cell non-Hodgkin lymphoma, e.g. lymphoplasmacytic lymphoma, PTCL NOS (Fig. 5.71) and especially ALCL (Fig. 5.72), can also have large nucleoli similar to Reed-Sternberg cells.^9,187 The distinction is particularly difficult in the presence of eosinophils, plasma cells and epithelioid cells, which is not unusual in T-cell lymphoma. Immunological studies may be necessary to solve the problem. There are some examples of large malignant cells with very large nucleoli and with many eosinophils and reactive lymphoid cells in the background, representing single malignant cells of metastatic – especially nasopharyngeal – carcinoma (Fig. 5.73), which were misdiagnosed as HL. In addition, megakaryocytes can be mistaken for Reed-Sternberg cells, e.g. in nodal myeloid metaplasia/extramedullary hematopoiesis. Also rarely with follicular dendritic cell sarcoma, Reed-Sternberg-like cells may also be seen in an inflammatory background (Fig. 5.74).

Fig. 5.69 Reed-Sternberg-like cells in infectious mononucleosis

(A) Multilobated giant cell (MGG, IP); (B) Binucleate cell with very large nucleoli (MGG, HP), from two different cases of infectious mononucleosis.

Fig. 5.70 Reed-Sternberg-like immunoblasts

(A) Large binucleate cell in case of angioimmunoblastic T-cell lymphoma; (B) Binucleate cell in toxoplasma lymphadenitis (MGG, HP).

Fig. 5.71 Reed-Sternberg-like cells in peripheral T-cell lymphoma

Pleomorphic T-cell lymphoma with Reed-Sternberg-like cells (MGG, IP).

(Reproduced with permission from van Heerde et al.⁹)

Fig. 5.72 Anaplastic large cell lymphoma

Large cell anaplastic lymphoma, CD30 positive; differential diagnosis of Hodgkin lymphoma is a common problem in ALK negative cases (MGG, LP).

Fig. 5.73 Reed-Sternberg-like cells in metastatic carcinoma

(A) Large binucleate cells with large nucleoli in metastatic nasopharyngeal carcinoma; note eosinophils in the background; (B) Large binucleate cell in lymph node metastasis from squamous cell carcinoma of floor of mouth (MGG, HP).

Fig. 5.74 Reed Sternberg-like cell in dendritic follicular cell sarcoma

Large binucleate cell with large nucleoli (Diff-Quik, HP oil).

The syncytial variant of HL, an uncommon form of nodular sclerosis HL, can be difficult to distinguish from NHL, carcinoma, melanoma, germ cell tumor and at times even sarcoma on FNB smears. Smears show cohesive sheets and clusters of large malignant cells with vesicular nuclei, prominent nucleoli and pale cytoplasm, and sometimes necrotic debris (see Fig. 5.68). Giant cells and multinucleated cells of Reed-Sternberg type can be recognized and may be numerous.¹⁸⁸ An immunologic panel can help in the diagnosis (see Table 5.3).

Clusters of epithelioid histiocytes are sometimes seen in smears of HL – as in some NHL – which could suggest granulomatous lymphadenitis (Fig. 5.75). The lymphoid cells must therefore always be carefully scrutinized in lymph node smears containing epithelioid cells.

Fig. 5.75 Hodgkin lymphoma

Clusters of epithelioid histiocytes resembling granulomatous lymphadenitis; note large abnormal cells lower right (MGG, HP).

Lymphocyte-rich smears may hide scanty numbers of large lymphoid neoplastic cells, resembling reactive lymphoid hyperplasia. Lymphocyte-rich classic HL may have a prominent small lymphocytic population with a few scattered Reed-Sternberg cells and variants. This may be difficult to distinguish from lymphocyte-predominant HL or T-cell/histiocyte rich B-cell lymphoma (see Fig. 5.46). Both of these may also have a paucity of large neoplastic cells in a rich reactive background. FNB is limited in making a distinction between nodular lymphocyte-predominant HL and T-cell/histiocyte rich B-cell lymphoma as the vital architecture cannot be assessed. To make the distinction between these entities, immunologic studies need to be carried out on a formal node biopsy.

Falling into the gray-zone lymphoma group are cases morphologically resembling classic HL; however, their immunophenotype is that of DLBCL ,NOS. A composite lymphoma needs to be excluded in biopsy specimens before making this classification.

FCM is of minimal value in the diagnosis of HL. An excess of CD4-positive T cells can be seen and a CD4/CD8 ratio greater than 4 should make one consider the diagnosis, even though it is not diagnostic. In nodular lymphocyte-predominant HL, not infrequently it may have a considerable proportion of CD4+CD8+(double-positive) T cells, a pointer to its diagnosis.⁶⁹