Mohamed Osman, MD, Hematopathology fellow
Nam Ku, MD, Assistant Clinical Professor UC Davis
Denis Dwyre, MD Professor UC Davis
Evaluation of a patient for a possible diagnosis of lymphoma requires a biopsy of a suspicious lymph node or other potentially involved tissue, along with the expertise of a pathologist. Evaluating a surgical specimen for lymphoma, commonly referred to as a lymphoma work-up, is a frequent request received by hematopathologists, specialists in the diagnosis of lymphomas and leukemia. The WHO classification of lymphoma is a tool of the hematopathologist used in the diagnosis and classification of lymphomas and leukemias. Detailed in the WHO, these diagnoses are typically based on the type of cell from which they are derived (mature and immature B-cells, T-cells, NK-cells, etc.), which is determined by the evaluation of the tissue by histologic morphology, immunophenotype, cytogenetics, molecular studies, along with clinical patient findings. Due to the necessity of multiple modalities for the diagnosis of lymphoma, these cases can be challenging for the pathologist.
Appropriate therapy for this malignancy requires a correct diagnosis. Many factors can affect the complete and accurate pathologic diagnosis of lymphoma. One important factor that can adversely hinder the diagnosis is the pathologist receiving a less than optimal specimen in the surgical pathology grossing room. Here we will discuss the optimal surgical specimen for a lymphoma workup and evaluation.
A typical lymphoma-workup consists of following components:
1. Tissue Microscopic Examination / Histology:
Morphologic evaluation of a formalin fixed, paraffin embedded tissue is essential for the proper classification of a lymphoma. Cytomorphologic features helpful in the evaluation of lymphoma include the cell size and nuclear features of the infiltrating tumor cells, as well as the architectural patterns. The background non-neoplastic components can also provide important diagnostic clues. Immunohistochemical (IHC) stains targeted against cellular proteins are typically utilized in order to characterize and phenotype the malignant cells. The stains can assist in evaluating the abnormal population identified by flow cytometry, or to screen reactive tissue to determine if a subtle abnormal population is present.
2. Flow cytometry:
Flow cytometry is an invaluable tool in diagnosing lymphoma. It provides a quick and sensitive mean to demonstrate an abnormal/clonal hematolymphoid population. The decision to use flow cytometry must be made before fixation because testing requires fresh tissue, typically suspended in a special nutrient medium, such as RPMI solution. Similar to IHC stains, flow cytometry can evaluate, characterize, and phenotype leukemias and lymphomas. Unlike IHC stains, flow cytometry most frequently evaluates only surface cellular proteins and does not assist in tissue architecture. Flow cytometry, however, can evaluate a larger number of cells than IHC staining of a tissue. Its utility may be limited in neoplasms where the number of tumor cells is limited, such as Classical Hodgkin lymphoma, non-hematologic malignancies, or in tissue samples with low recoverable cell counts/low viability.
3. Cytogenetics and Molecular Testing:
Tumor cytogenetics, or karyotyping, can be helpful in classifying some lymphomas. Certain lymphomas have characteristic chromosomal translocations that can aid the pathologist in the diagnosis of lymphoma. Occasionally chromosomal abnormalities can also provide prognostic information as well. Fresh tissue is required for a conventional karyotype. Certain abnormalities, such as balanced translocation, rearrangements, or hyper/hypodiploidy can also be detected by interphase Fluorescent In-Situ Hybridization (FISH) studies using a formalin-fixed paraffin embedded (FFPE) tissue. Sometimes small deletions (such as del17p) are difficult to detect in fixed tissue. Other molecular studies, such as PCR testing and next generation sequencing, can also be helpful in the diagnosis and classification of lymphomas. For example, gene rearrangement studies can assist in demonstrating clonality for the determination of lymphoid malignancies.
Laboratory Best Practice: Optimal Specimen for Adequate Lymphoma Workup
Lymph node sampling can be achieved by different methods including a fine needle aspiration (FNA), an image-guided core needle biopsy, or by an excisional biopsy.
As the classification of lymphoid neoplasms is based on all available information to define disease entities, having sufficient tissue for this multiparameter approach is critical. Thus, FNA alone is often not adequate in the amount of tissue for initial diagnosis and generally not recommended by pathologists. FNA’s typically do not provide sufficient tissue for histology with IHC staining and a complete flow cytometry panel. A core needle biopsy provides more tissue, but is not always optimal either due to a limited amount of tissue. Studies have shown that 25-35% of FNA/core needle biopsies require follow-up excisional biopsies for full classification. 
A workshop at the 11th International Conference on Malignant Lymphoma in Lugano, Switzerland, in June 2011 that included leading hematologists, oncologists, radiation oncologists, pathologists, radiologists, and nuclear medicine physicians, representing major international lymphoma clinical trials groups and cancer centers, recommended that an incisional or excisional biopsy is preferred, and a core-needle biopsy can be considered when excisional biopsy is not possible due to location of the mass or other clinical reasons. National Comprehensive Cancer Network (NCCN) also recommends an incisional or excisional biopsy to establish the diagnosis of lymphoid neoplasms.
So why is an excisional biopsy better?
Provides ample tissue for all necessary testing.
Allows evaluation of architectural patterns and accurate grading.
Reduces sampling errors that can be seen in paucicellular neoplasms or partial involvement.
Eliminates the need for additional biopsies and possible delay in treatment.
It is our recommendation that the treating physicians provide excisional biopsies whenever medically possible in order for the pathologist to provide a complete as possible diagnosis in order to provide the best care for the patient.
- Swerdlow SH, Campo E, Harris NL, Pileri SA, Stein H, Thiele J (Eds): WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (Revised 4th edition) IARC:Lyon 2017
- Gabriel Caponetti, MD, and Adam Bagg, MD, JCSO 2017;15(1):43-48. ©2017 Frontline Medical Communications. doi: https://doi.org/10.12788/jcso.0328.
- Russell A. Higgins, Jennifer E. Blankenship, and Marsha C. Kinney (2008) Application of Immunohistochemistry in the Diagnosis of Non-Hodgkin and Hodgkin Lymphoma. Archives of Pathology & Laboratory Medicine: March 2008, Vol. 132, No. 3, pp. 441-461.
- Frederiksen, John K., et al. “Systematic review of the effectiveness of fine-needle aspiration and/or core needle biopsy for subclassifying lymphoma.” Archives of Pathology and Laboratory Medicine2 (2015): 245-251.
- Recommendation for Initial Evaluation, Staging, and response Assessment of Hodgkin and Non-Hodgkin Lymphoma: The Lugano classification. Bruce D. Cheson, Georgetown University Hospital, Lombardi Comprehensive Cancer Center, Washington, DC; Richard I. Fisher, Fox Chase Cancer Center, Philadelphia, PA; Sally F. Barrington, St Thomas’ Hospital; T. Andrew Lister, St Bartholomew’s Hospital, London, United Kingdom; Franco Cavalli and Emanuele Zucca, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; and Lawrence H. Schwartz, Columbia University, New York, NY.