Establishing the Diagnosis of Lymphoma: From Initial Biopsy to Clinical Staging

Introduction

During the past decade, a significant amount of clarity has been brought to our understanding of the pathogenesis of non-Hodgkin’s lymphoma. Many of the insights gleaned from our knowledge of the molecular mechanisms that underlie lymphomagenesis have translated into improved classification schemes. The Working Formulation is a clinically driven scheme that has long since outlived its usefulness.[1] It has largely been replaced by the Revised European and American Lymphoma classification, now known as the REAL proposal.[2]

This scheme recognizes “real” disease entities that have a clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic correlate. Within each lymphoma category, a spectrum of clinical behavior may be seen, and this diversity is important to our understanding of non-Hodgkin’s lymphoma.[3]

Pathologists welcome this classification, as it focuses on the biology of these disorders rather than lumping diseases together based on a common clinical outcome. The information imparted by an accurate diagnosis, together with the relevant clinical data, can now be used to make definitive treatment decisions.

Classification Techniques

The newly developed and more sophisticated techniques for analysis of lymphoma cells have provided us with the tools necessary for precise classification of non-Hodgkin’s lymphoma. Nonetheless, routine histologic studies remain the gold standard for diagnosis. This review will focus on the approaches used both for diagnosis and staging and will attempt to provide some guidelines as to how and when these tests should be employed.

Excisional Biopsy

A well-processed hematoxylin and eosin (H&E) stained section of an excised lymph node is the mainstay of pathologic diagnosis.[4] Most often, the diagnosis of difficult lesions relies heavily on a careful assessment of the underlying architecture. Lymphoma diagnoses are much less about cytologic detail and far more about altered architecture. For example, follicular small-cleaved cell lymphoma (FSC) is characterized by an abundance of neoplastic lymphoid follicles containing monomorphous small-cleaved lymphocytes. The individual cells themselves, however, are otherwise typical smallcleaved lymphocytes seen in the benign follicles of reactive lymph nodes.

The loss of normal nodal architecture that accompanies an infiltrate is of paramount importance in making a diagnosis. An incisional lymph node provides only a glimpse of the architecture, making interpretation difficult. Our surgical colleagues must be instructed to biopsy the most clinically significant site, and whenever possible, to remove an intact lymph node for pathological processing. The tissue should be delivered fresh to pathology at an appropriate time of the day in order to maximize the material for lymphoma protocol studies.

Many hematopathologists prefer to triage the material using imprint preparations, whereby a fresh cut surface of the node is touched onto glass slides for Romanowsky staining. Experienced pathologists are able to make a good approximation of the disease process based on the touch prep morphology, thus resulting in the efficient ordering of additional tests.

When the size of the tissue is limiting, the first priority must be to process the material routinely for fixation and H&E sections. Properly fixed specimens can be used for regular histologic examination, paraffin(Drug information on paraffin) section immunoperoxidase staining, and depending on the fixative, for gene rearrangement studies by polymerase chain reaction (PCR).[5] Although B5 is the optimal fixative for routine lymphoid histology and is preferred for immunoperoxidase studies, it precludes PCR studies in most laboratories. Formalin fixation is preferred when the biopsy is small because all of the above studies, including PCR, can be performed.

Diagnosing Disease at Extranodal Sites—Approximately 30% to 35% of cases of non-Hodgkin’s lymphoma in adults present primarily at extranodal sites. Much less is known about the molecular mechanisms involved in these disorders in comparison to node-based disease. Therefore, it is important to remember to process extranodal biopsy material for lymphoma protocol studies whenever there is a suspicion of a hematolymphoid neoplasm.

Molecular genetic and cytogenetic data from gastric and pulmonary resection specimens have enormous potential to provide insights into the pathogenesis of mucosal-associated lymphoid tissue (MALT) lymphomas but, unfortunately, lymphoma protocol is frequently overlooked in this setting.[7,8] Nonetheless, examination of a well-processed H&E section from an excisional biopsy by an experienced hematopathologist will be sufficient to establish a diagnosis in the majority of cases.

Needle-Core Biopsy

Needle-core biopsies have a role in lymphoma pathology, although it remains limited.[9] The use of 14 to 22 gauge needles under ultrasound or radiological guidance to establish a diagnosis of non-Hodgkin’s lymphoma is problematic because of technical difficulties with biopsy crush artifact, inadequate sampling, and the usual vagaries of lymphoma pathology. Although this technique has advantages over fine-needle aspiration (FNA), it should be used judiciously as a diagnostic tool for patients with suspected non-Hodgkin’s lymphoma. Needle-core biopsies do allow a minimal assessment of architecture in addition to immunostaining procedures, but interpretation can be problematic in cases of T-cell rich B-cell lymphoma, angioimmunoblastic-type peripheral T-cell lymphoma, or MALT lymphoma where much of the lymphoid infiltrate is reactive.

A careful review of most excisional lymph node biopsies demonstrates marked cytologic and architectural variation throughout the section, underscoring the complexity of non-Hodgkin’s lymphoma diagnoses in what would otherwise be considered routine circumstances. Needle-core biopsies are unable to detect this variability, leading to the possibility of incorrect diagnoses in many cases. Although recent studies have recommended increased use of these techniques, patient selection and failure to provide convincing evidence that the “right treatment” decision was made in the majority of cases hamper their interpretation.[10,11] Also, many of these studies included patients with an established diagnosis of either non-Hodgkin’s lymphoma or Hodgkin’s disease—an approach that differs significantly from a diagnostic procedure.[9]

In managing ill patients or those with significant comorbid disease who are unable to tolerate an invasive surgical procedure, needle-core biopsies offer a better alternative to FNA for the diagnosis of intra-abdominal or thoracic disease. Ideally, two or three cores should be obtained with one core routinely processed for histology and the remainder used for lineage and clonality studies. In this setting, cautious interpretation of the biopsy by an experienced hematopathologist and integration of the results of the ancillary studies should allow a reasonable treatment decision to be made in most cases.