The diagnosis of non-Hodgkin lymphoma using fine-needle aspiration cytopathology

A work in progress


  • Paul E. Wakely Jr MD

    Corresponding author
    1. Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio
    • Department of Pathology, Ohio State University College of Medicine, 414 Doan Hall/410 West 10th Avenue, Columbus, OH 43210
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The utility of fine-needle aspiration biopsy in patients with non-Hodgkin lymphoma remains unsettled. Competing philosophies continue to debate its proper role in this cancer.

Enlarged lymph nodes are among the most common sites encountered in an active fine-needle aspiration (FNA) cytopathology practice. In adults, many of these lymph nodes harbor metastatic deposits, which FNA biopsy is well-positioned to diagnose with relative ease and accuracy,1 and functions as a readily accepted form of clinical management. However, some examples of lymphadenopathy are secondary to various forms of malignant lymphoma. A large body of literature exists emphasizing the significance of FNA for patients with non-Hodgkin lymphoma (NHL). Nevertheless, its proper role remains a contentious matter. This is in part because of perceived “turf” issues with hematopathologists, but is also a consequence of markedly divergent opinions regarding the utility (or lack thereof) of FNA cytology. A single, but influential, study demonstrated the unfortunate reality of diagnostic accuracy in the United States when lymphoma diagnosis is attempted without the benefit of many of the components required for not only definitive subclassification, but merely the recognition of lymphoma using FNA cytology.2 This study demonstrated no real benefit from FNA. Nevertheless, immunophenotyping (IP) was performed in <50% of cases in that study, and only approximately one-third of cases had diagnoses that conformed to an accepted lymphoma classification system. This publication correctly concluded that if approached in this manner, the FNA procedure for lymphoma diagnosis is “woefully inadequate” and potentially misleading.

Others have asserted that FNA is so accurate in the diagnosis and subclassification of NHL that histologic evaluation will most likely cease to be necessary for the diagnosis and follow-up of patients with NHL, thereby avoiding useless and costly surgical biopsies.3 Conclusions such as these, and lectures questioning the need for histopathology4 have in my view overstated the diagnostic performance of FNA in patients with NHL, and are examples of unfettered hubris. These claims also have led to friction with our hematopathology colleagues, some of whom have criticized us for being “married to a technique.”

The proper role of FNA in NHL is highly dependent on the interest and expertise of those practitioners applying FNA to this category of neoplasms. For advocates of the FNA form of biopsy (myself included), one's enthusiasm must be tempered with evidence-based results. The following question has been asked: can and should the diagnosis of lymphoma be made on FNA cytology (even with flow cytometry [FC]) and, if so, what is gained by this?5 Numerous publications have answered parts of this question in the affirmative. A few more challenging questions include: 1) Can FNA cytopathology not only diagnose NHL but also correctly subclassify it (particularly as an initial diagnosis), and do this on a regular, routine basis? 2) Is the more proper role of FNA to serve as a triage tool for NHL patients? and 3) Should the cytologic diagnosis of NHL be transferred in part or fully to hematopathologists, or must all cases be evaluated by both subspecialties before a diagnosis is issued? A consensus opinion among pathologists to these queries remains unresolved. Most likely, the most controversial aspect surrounding the use of FNA in NHL is whether a patient's initial diagnosis of NHL should rest solely on an FNA interpretation. Current practice in most centers in the United States dictates an excisional biopsy if the lymph node is surgically accessible; however, for deeply situated lymph nodes/masses (eg, those that are intra-abdominal or intrathoracic), an initial FNA diagnosis may stand alone depending on the patient's comorbidities and the type of intervention required.

Table 1 lists some of the principal obstacles hindering the routine use of FNA in the diagnosis and subtyping of most examples of NHL. These impediments apply particularly to those instances in which an original diagnosis of NHL is attempted. The most conspicuous reason behind the difficulty of an FNA-based specific NHL diagnosis (compared with an FNA-based diagnosis of metastatic carcinoma to lymph node) is the inherent complexity of past and current NHL classifications. Compounding this complexity is a continually escalating list of malignant hematolymphoid subtypes, which is overwhelming for most non-hematopathologists. The latest (2008) iteration of the World Health Organization (WHO) classification of lymphomas6 demonstrates that the number of nonleukemic, mature B-cell lymphoma subtypes alone has nearly doubled when compared with its classification from merely 7 years earlier.7 As an example of this complexity, the distinction of grade 3A follicular lymphoma (FL) from grade 3B FL is currently emphasized because 3B is believed to share more similarities with diffuse large B-cell lymphoma (DLBL). To distinguish large confluent follicles of grade 3 FL from DLBL, the WHO states that it may be essential to stain for follicular dendritic cells,6 a difficult if not impossible task to say the least with an FNA specimen.

Table 1. Some Impediments to FNA Biopsy as a First-Line or Sole Approach to NHL Diagnosis
  1. FNA indicates fine-needle aspiration; NHL, non-Hodgkin lymphoma.

• Inherent and ever-expanding complexity of NHL classification
• Lack of cytopathologist in-depth knowledge or even interest in NHL classification and its nuances
• Immediate assessment of adequacy logistically difficult in many practices
• Routine requirement for ancillary testing in nearly all cases
• Ease of surgical resection of an entire lymph node in most presumed NHL cases
• Requirement for hematopathologist input
• Loss of archival material for research/retrospective analysis

Oncologists rightfully demand as specific an NHL diagnosis as possible using the most up-to-date classification if patient management is to be based on an FNA diagnosis of NHL alone. Anything less will force the performance of an open biopsy, most especially when the patient presents with no prior history, and superficial lymphadenopathy makes excisional biopsy easily accessible. This demand for an open biopsy lessens as the surgical risk necessary to acquire sufficient tissue for an exact diagnosis increases and tissue becomes more difficult to procure. Clinically unstable patients and those with deep-seated masses (retroperitoneal, intra-abdominal, pelvic, mediastinal, and pulmonary masses) will definitely benefit from FNA biopsy, but whether FNA carries an advantage over even core needle biopsy (CNB) is still debatable.

As a corollary to the first impediment shown in Table 1, many cytopathologists are just not familiar enough or fluent with the nuances and intricacies of the various subtypes of NHL. This is in part because lymphoma classifications continue to be updated and revised on a more frequent basis compared with other tumor classifications. A cytopathologist must be highly motivated to remain abreast of these developments. As stated by Safley et al, the significant cytologic overlap and IP heterogeneity among different subtypes of B-cell NHL have impacted the broader implementation of FNA in the subclassification of NHL, and reduced clinicians' enthusiasm for implementing FNA as a complete modality in the initial diagnosis of patients with lymphoma.8 It goes without saying that it is much easier to sign out an FNA biopsy in noncommittal diagnostic phrases such as “atypical lymphoid infiltrate,” “probable lymphoma,” “suspicious for lymphoma,” “malignant lymphoma,” or even “B-cell lymphoma” (which is possible with IP of the aspirate, but still insufficient for treatment in the majority of new patients), and recommend an excisional biopsy for definitive diagnosis and classification. In many centers, both oncologists and cytopathologists are completely satisfied with this algorithm. In those centers in which a desire to alter this approach, and have FNA be a “complete modality” in the diagnosis of initial or recurrent NHL, exists, cytopathologists must take a real interest in hematopathology. It cannot be overemphasized that a team approach in which hematopathologist colleagues are involved in these cases is essential. Anything less will have difficulty succeeding.

In the United States, nearly all examples of NHL require ancillary testing before a definitive diagnosis is rendered. This is certainly the case in patients without a history of any form of lymphoma. Thus, light microscopic evaluation made from Romanowsky, Papanicolaou, or hematoxylin and eosin-stained smears is merely only the beginning of the diagnostic process, which often requires the pathologist to answer prognostic and treatment questions. The workup routinely entails a multiparametric approach including at a minimum IP, but may also necessitate fluorescence in situ hybridization (FISH), polymerase chain reaction, and paraffin-embedded cell block preparation. The majority of IP is currently performed using 4-color FC, but as all hematopathologists are aware, FC is not an infallible technique. For example, a significant percentage of large cell lymphomas are not analyzable using FC because of poor cell viability, even in the best of laboratories. Either aberrant expression or a lack of certain surface markers may lead to confusion and an inability to specifically subtype some lymphomas. This could lead to misdiagnosis if there is over-reliance on IP to classify some B-cell lymphomas.9 Recall that certain lymphomas demonstrate lineage plasticity lacking a consistent, specific immunoprofile, and FC also has no utility in the analysis of surface immunoglobulin-negative lymphomas. All this testing necessitates extracting a sufficient number of cells to perform an immediate assessment of the aspirate to assure cell adequacy (without crush artifact) and a systematic, disciplined protocol for specimen handling. This requires more time and effort, and the proper handling of an aspirate than many centers currently provide. Many hospitals/medical centers do not even possess the personnel to offer an onsite adequacy assessment service let alone ensure the correct handling of the aspirate. As was made unmistakably clear in the article by Hehn et al, if only 38% of aspirates from laboratories in 21 different cities have IP performed, the desire or obligation to pursue a specific diagnosis of NHL (let alone any attempt to subtype it) using FNA does not exist in the minds of many, if not most, pathologists in the United States. The result therefore is a dismal correlation with tissue diagnoses.2

A decade ago, I reviewed 30 random articles regarding FNA of NHL from the English literature (using no specific criteria to choose those articles), concentrating on articles published during the last decade of the 20th century.10 Conclusions from that search demonstrated an enormous range in diagnostic sensitivity, but specificity in a particular laboratory's ability to exclude malignant lymphoma was >90% for all but 2 articles. The division of NHL into specific subtypes attempted in nearly all of those articles demonstrated that subclassification was correct in approximately two-thirds of the articles, most in patients with recurrent disease.

I have repeated that exercise in examining another 30 articles published in the first decade of this century (Table 2).3, 8, 11-38 This time, criteria for inclusion included that a minimum of 10 cases were studied, sensitivity and specificity were either clearly stated or gleaned from the results, the authors made a firm diagnosis of lymphoma (not suspicious for lymphoma or atypical lymphoid cells) in determining sensitivity, and the Revised European-American Classification (REAL) or 2001 WHO classification was used when subclassification was attempted. Table 2 is not, nor is it meant to be, an all-inclusive list. There was no conscious attempt at selection bias and no attempt to separate accuracy based on primary diagnosis versus recurrent disease, and I did not take into account the percentage of aspirates considered nondiagnostic or insufficient for diagnosis. Because of the marked variation in the selection of cases among these 30 publications, no meta-analysis of these data are possible. Nevertheless, Table 2 allows one to roughly conclude that the major strength of FNA in this malignancy is its specificity, even in extranodal sites. Sensitivity in recognizing NHL varied from 0% to 100%, whereas the mean sensitivity was 78%. Not all aspirates diagnosed as NHL underwent follow-up tissue examination to confirm that diagnosis. Note that IP was performed in all but 2 (93%) of these articles; however, closer examination of each article indicated that IP was not performed in all cases in many of these studies. Accuracy in NHL subclassification, attempted in only a minority (43%) of articles in this most recent analysis, demonstrated no improvement, even with access to the more recent molecular tools at our disposal within these past 10 years8, 31 and application of the “cytopathology-friendly” 2001 REAL/WHO classification. In 13 publications in which it was stated, approximately two-thirds (72%) of NHL cases were accurately subclassified, which is similar to the figure published earlier.10 Several of these studies demonstrated broad variations in the level of difficulty in subtyping different forms of NHL. There was high accuracy in the recognition of small lymphocytic lymphoma by some,11, 14 but dismal results for marginal zone lymphoma11, 14, 28 and some T-cell lymphomas.33, 36 In examining these results, one must also be aware of the potential for publication bias, a phenomenon whereby significant results are more apt to be submitted for publication than insignificant or negative findings.39

Table 2. Results From FNAB of NHL, 2000 Through 2010
StudyNo. of NHL Only CasesIP?Sensitivity, %Specificity, %Correctly Subclassified, %Comments
  • FNAB indicates fine-needle aspiration biopsy; NHL, non-Hodgkin lymphoma; IP, immunophenotyping; FC, flow cytometry; IHC, immunohistochemistry on cell block; ND, not done; PTCL, peripheral T-cell lymphoma; BCL, B-cell lymphoma; EUS-FNA, endoscopic ultrasound-guided fine needle aspiration; MZL, marginal zone lymphoma; FISH, fluorescent in situ hybridization; MtCL, mantle cell lymphoma; PCR, polymerase chain reaction; ALCL, anaplastic large cell lymphoma; AILT, angioimmunoblastic T-cell lymphoma.

  • a

    Subclassification on small and medium-sized NHL only.

  • b

    Only 42% of primary lymphomas were subclassified correctly.

  • c

    Only a percentage of cases were subclassified.

Chhieng 20011156Yes/FC, IHC8210085Small cell NHL cases only
Meda 200012155Yes/FC8585ND 
Nicol 20001381Yes/FC90100ND 
Dong 200114139Yes/FC6710077 
Liu 200115127Yes/FC8910077 
Yao 20011633Yes/FC, IHC9110077PTCL cases only
Mourad 20031774Yes/FC10010063 
Zeppa 20043160Yes/FC9210061a 
Mayall 20031825Yes/FC, IHC48100NDUncommon forms of BCL
Nasuti 20001948Yes/FC, IHC9294ND 
Dey 20062048Yes/FC9010079 
Mehra 20052110Yes/FC73100NDEUS-FNA only
Ribeiro 20012222Yes/FC7493NDEUS-FNA only
Al-Haddad 20092338Yes/FC8092NDEUS-FNA only
Sangalli 20012417Yes/IHC59100NDThyroid FNA only
Cha 20022512No58100NDThyroid FNA only
Lerma 20032612No92100NDThyroid FNA only
Gupta 20052710Yes/IHC90100NDThyroid FNA only
Crapanzano 20032814Yes/FC, IHC36100NDMZL cases only
Levine 20042919Yes/FC, IHC10010068bBreast FNA only
Wakely 20013014Yes/FC, IHC78100NDSoft tissue FNA only
Bentz 20043110Yes/FC, FISH100100100All MtCL cases
Caraway 20053255Yes/FC, FISH951008031% MtCL cases
Safley 2004822Yes/FC, FISH, PCR10010064 
Rapkiewicz 20073320Yes/FC, IHC7010035ALCL cases only
Zhang 20103415Yes/FC, FISH, PCR73100ND 
Monaco 200935181Yes/FC, FISH94100NDcBCL cases only
Ng 20023616Yes, IHC00NDAILT cases only
Khashab 20103716Yes, FC7710069Pancreatic FNA only
Chhieng 20003818Yes, FC77100NDSalivary gland FNA only

FNA biopsy functions as an extremely valuable clinical tool in the diagnosis and management of patients with recurrent lymphoma, particularly when coupled with IP. However, its current usage and goal (at least in most US medical centers) remain somewhat modest, but nonetheless valuable in a patient harboring an undiagnosed NHL. For these individuals, the ability to distinguish NHL correctly from a nodal or extranodal FNA appears to suffice for many oncologists and pathologists. Further subclassification is typically deferred to a surgically resected specimen.

A major exception to this practice occurs in those limited number of centers in which standardized protocols for cell collection/processing are adhered to, in which every attempt is made to correctly subtype an NHL aspirate, and in which pathologists who are also cognoscenti in the field of hematopathology staff the cytology laboratory. One approach to potentially compensate for this lack of such high standards based on using an FNA-only approach is to add CNB in tandem with FNA. As in many other anatomic sites (eg, thyroid, soft tissue, kidney), CNB has been advocated as either a supplement to or replacement for FNA. Mixed results appear to moderate the impact of CNB in the diagnosis of lymphoma. In a study of 101 patients, Lachar et al claimed a sensitivity of 90% for establishing a diagnosis of lymphoma, and 95% for correctly classifying lymphoma.40 However, their study had <5% of patients undergo excisional biopsies with which to compare these results because treatment was administered based on the CNB diagnosis alone. A study of only 28 patients restricted to those with deep abdominal lesions demonstrated an increase in accuracy from 82% to 93% when CNB was combined with FNA and FC; however, this increase is not statistically significant.41 Gong et al concluded there was no clear advantage to the diagnosis and classification of small B-cell NHL by adding CNB to FNA, but it was a useful adjunct in the diagnosis of DLBL.42

For FNA biopsy to advance in the United States beyond its contemporary role as a screening tool in many (but not all) new NHL patients requires at a minimum several developments. These include a critical mass of cytopathologists who are willing to develop a thorough understanding of the most recent WHO classification of lymphomas (recently updated),6 who are able to collect sufficient cells for IP (and often other studies), and who develop close interactions with their hematopathologist colleagues. I believe these substantive demands are achievable; however, as it currently stands, the diagnosis of NHL using FNA cytopathology remains a work in progress.


The author made no disclosures.