Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
ABSTRACT
The lymph node plays an important role in the lymphatic spread of abnormal antigens from exogenous or endogenous sources, including infectious agents, foreign bodies, self-antigens, and malignant cells, by harboring various immune cells that react to abnormal antigens and their sources. This often leads to enlargement of the lymph node, also known as “lymphadenopathy.” In this review article, malignancy of the lymph node is the main focus, especially regarding how general practitioners and pathologists can achieve a definitive diagnosis. The basic principle relies on the normal structure, cellular components, and functions of the lymph node as well as the types of malignancy found. Careful clinical history taking of any possible cause of lymphadenopathy warrants exclusion of any mimics of malignancy of the lymph node, including drug reactions and immunodeficiency states. An adequate cell or tissue sample allows pathologists to work efficiently by mastering the multimodality approach under good clinical collaboration. Effective communication between pathologists and physicians regarding relevant laboratory investigations should make it easier to diagnose a specific type of malignancy. This review article also focuses on how general pathologists handle cell or tissue samples by conventional morphologic evaluation and panels of immunohistochemistry so that general practitioners understand the diagnostic process and understand how to diagnose malignancy of the lymph node.
INTRODUCTION
Malignancy of the lymph node can be divided into primary (the cellular components) and secondary or metastatic tumor, including leukemic infiltration. A primary malignancy of the lymph node is mostly the result of neoplastic lymphoid cell clone, best known as malignant lymphoma. Meanwhile, secondary malignancy is usually a metastatic tumor of nearby structures or at times an unknown primary site.1 Leukemic infiltration of the lymph node is also a secondary malignancy, but terminology prefers to use infiltration (or involvement) to
metastasis. However, this is uncommon prior to typical leukemic manifestation in general.2 Leukemic infiltration, poorly-differentiated neuroendocrine carcinoma (“small cell carcinoma”), metastatic invasive lobular carcinoma, and other metastatic tumors may create difficulties in making a definitive diagnosis because they share morphologic similarities to malignant lymphomas. This particular group of tumor cells with small round cell morphology is often referred to as “small round-cell tumor (SRCT),3,4 however, pathologists should pay close attention to the size of tumor cells because after provisional diagnosis,
Corresponding author: Sanya Sukpanichnant E-mail: sanya.suk@mahidol.ac.th
Received 17 July 2022 Revised 4 August 2022 Accepted 4 August 2022 ORCID ID: http://orcid.org/0000-0002-9724-2692 http://dx.doi.org/10.33192/Smj.2022.72
All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.
immunohistochemistry (immunostaining) can reveal diffuse large B-cell lymphoma (DLBCL). In such cases, discrepancy between provisional diagnosis of SRCT and final diagnosis of DLBCL can lead to a concern about the proficiency of the pathologist. In order to provide a definitive diagnosis of a malignancy in the lymph node, general practitioners and pathologists should understand the following topics.
The lymph node plays an important role in handling the lymphatic spread of abnormal antigens from exogenous or endogenous sources, including infectious agents, foreign bodies, self-antigens, and malignant cells, by harboring various types of immune cells that react to abnormal antigens. The process usually leads to enlargement of the lymph node, also known as “lymphadenopathy.” Therefore, knowledge of the basic principles of normal structure, cellular components, and functions of the lymph node, as well as the types of malignancy commonly found in the lymph node should allow general practitioners and pathologists to make a definitive diagnosis.
Regarding primary or secondary malignancy of the lymph node, the normal structure of the lymph node can explain why tumor cells localized in lymph node sinuses tend to be metastatic tumors with lymphatic
spreading. In addition, some lymphoma cells can have a lymph node sinus distribution mimicking metastatic tumor. When tumor cells are localized in the paracortex (interfollicular area or the area of the lymph node between the cortex and medulla), it can be suggestive of primary or secondary malignancy of the lymph node. Normally, the paracortex is predominantly occupied by T-cells (the “T-cell zone”) but it is also the place where circulating cells enter the lymph node via postcapillary venules, commonly known as “high endothelial venules” (HEV) in immunology. Thus, not only T-cell lymphoma and B-cell lymphoma arising from a non-germinal center B-cell clone that are common in the paracortex, but also metastatic tumor cells with hematogenous spreading that can enter the lymph node via HEV in the paracortex. Certainly, leukemic cells can also reach the lymph node and cause lymphadenopathy via this route. The cortex, mostly occupied by B-cells and lymphoid follicles, is affected by malignant lymphomas, especially follicular lymphoma or other lymphomas arising from germinal center B-cells. Pathologists should be aware of the normal structure of the lymph node, especially the compartments commonly affected by various types of malignancy. The main challenge during microscopic examination of the lymph node is when tumor cells destroy normal structures of the lymph node (complete effacement of the lymph node) and the aforementioned clues cannot be used, leaving only morphologic evaluation of tumor cells as a possible solution.1,5 (Table 1)
TABLE 1. Compartments of the lymph node and possible types of malignancy.
Compartment | Common malignancy | Less common malignancy |
Lymph node sinus | Metastatic tumor with | Anaplastic large cell lymphoma, |
lymphatic spreading | rare variant of diffuse large B-cell | |
lymphoma, and Langerhans cell | ||
histiocytosis | ||
Paracortex (interfollicular area) | T-cell lymphoma | B-cell lymphoma, metastatic tumor |
with hematogenous spreading, and | ||
leukemic infiltration | ||
Cortex | Follicular lymphoma and | Nodular T-cell lymphoma |
others arising from germinal | (arising from T follicular helper cells) | |
center B-cells | and follicular dendritic cell sarcoma | |
All compartments (diffuse | Any type of malignancy; definitive | diagnosis depends on morphologic |
effacement of the lymph node) features and immunophenotypic findings of the tumor cells | ||
Cellular components of the lymph node are important for understanding why some types of malignancy occur more frequently. The lymph node cortex is predominantly a B-cell zone, while the paracortex is an T-cell zone. The medullary cords are predominantly occupied by plasma cells while the medullary and subcapsular lymph node sinuses are occupied by sinus histiocytes. Also, follicular dendritic cells mainly occupy the germinal centers of the lymphoid follicles. Less common in the lymph node are plasmacytoid dendritic cells and interdigitating reticulum cells. These cellular components of the lymph node work together to react to incoming antigens via lymphatics or HEV. The antigens arrive at the lymph node via lymphatics and trigger proliferation of sinus histiocytes so that the lymph node sinuses are dilated via accumulation of sinus histiocytes. At this point, without any detectable tumor cells, the enlarged lymph node caused by sinus histiocyte hyperplasia (or “sinus hyperplasia”) is not diagnosed to have metastatic tumor.1,5 Pathologists should avoid the terminology “sinus histiocytosis” because a general practitioner can get confused with “sinus histiocytosis with massive lymphadenopathy (SHML)” or “Rosai-Dorfman disease (RDD).” The author once found a patient who unfortunately received a course of local irradiation for an enlarged lymph node diagnosed as “sinus histiocytosis” because the radiotherapist misunderstood that it was “SHML” or “RDD.” Fortunately, the patient was referred to Siriraj Hospital, the histologic slides were reviewed and a diagnosis of “sinus hyperplasia” of the lymph node was given instead of “sinus histiocytosis” to avoid misunderstanding as “SHML” or “RDD.” Sometimes, lymphoma in a lymph node dissection specimen from patients who underwent tumor resection are overlooked as pathologists generally pay attention to metastatic tumors in lymph node sinuses.6
The function of the lymph node in terms of immune reaction either helps or hampers diagnosis of malignancy. The most common immune reaction to tumor cells is involving tumor infiltrating lymphocytes (TIL), first described in malignant melanoma.7 Therefore, assessment of TIL has been proposed in other types of malignancy as well.8,9 In terms of morphologic evaluation of malignancy in the lymph node, TIL may lead to histologic features like lymphoepithelial carcinoma, indolent lymphoma, or T-cell rich variants in large cell lymphoma or classic Hodgkin lymphoma (CHL),5 depending on the size, number, and morphology of tumor cells.
Epithelioid histiocytes (a pathology term) or activated macrophages (immunology term) sometimes intermingle with tumor and other immune cells in the lymph node. They can form tiny clusters, aggregates, sheets, or even granulomas, that lead to the wrong diagnosis of granulomatous lymphadenitis. In some places, where infectious diseases are common, coexisting tuberculosis and malignancy are found in the same lymph node.10 When a lymph node with more clusters or aggregates of epithelioid histiocytes mixed with tumor cells, it can lead to the wrong diagnosis of granulomatous lymphadenitis. Therefore, pathologists should be aware of some tumors that have accompanying epithelioid histiocytes in clusters, aggregates, sheets, or even granuloma – germ cell tumor, CHL, and non-Hodgkin lymphoma (NHL) such as lymphoepithelioid lymphoma (“Lennert lymphoma”), a variant of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, T-cell/histiocyte rich large B-cell lymphoma, Burkitt lymphoma, and small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL).5,11,12 Lastly, aggregates or sheets of epithelioid histiocytes can mimic metastatic carcinoma at times.
Tumor necrosis in the lymph node is occasionally seen with or without any preceding history of fine needle aspiration of the lymph node. At times, lymph node infarction can also occur. Careful evaluation for vascular occlusion can reveal tumor emboli or angiodestruction by tumor cells; the latter is more common in NHL.5,13
Malignant lymphomas and metastatic tumors are classified as primary or secondary malignancy, respectively. Malignant lymphomas at present are defined as a malignancy of lymphoid cells, which is different than in the past when it was a malignancy of the lymphoid tissue. Therefore, in the present classification of malignant lymphomas, we do not have histiocytic lymphoma, which was common in the 1960s according to the Rappaport classification for NHL. The different types of malignant lymphoma require clinical, morphologic, immunophenotypic, and genetic findings in order to make a definitive diagnosis according to the revised 4th edition of the WHO classification, published in 2017.5 Malignant lymphoma can be divided into 3 types – B-cell lymphoma, T/NK cell lymphoma, and CHL – after excluding post-transplant lymphoproliferative disorder (LPD) and other iatrogenic immunodeficiency- associated LPD that can lead to varied pathologic findings, including any type of malignant lymphoma. A careful and complete clinical history review for immnosuppression is important in handling lymphadenopathy. Discontinuation of methotrexate or other immunosuppressive drugs can lead to improvement in clinical outcomes within one
week and spontaneous regression of the enlarged lymph node within three weeks.5,14
For general practitioners and pathologists, malignant lymphoma can be perplexing with 51 established entities plus six provisional entities of malignant lymphoma, according to the 2017 WHO classification.5 A simplified
version is presented in Table 2 with an emphasis on important issues that general practitioners and pathologists can help to manage efficiently for the lymphoma patients. Regarding secondary malignancy of the lymph node, metastatic tumors are more common than leukemic infiltration or plasma cell myeloma (multiple myeloma).
TABLE 2. A simplified classification of malignant lymphomas for general practitioners and pathologists (modified from revised 4th edition of WHO classification, 2017)5
Based on historical approach: | Based on pattern: nodular vs diffuse | B-cell lymphoma - B-LBL | - LPL | IGH gene, kappa Ig gene, lambda Ig gene |
Hodgkin vs Non-Hodgkin | BL: MYC DLBCL: MYC; BCL2; BCL6; IRF4; MYD88; FL: BCL2 MCL: CCND1 MZL: BIRC3 (API2), MALT1, BCL10 | |||
Based on clinical | Based on cell size: | T-cell lymphoma & NK/T-cell | TCR genes | |
behavior: | small vs large (including | lymphoma | AITL: RHOA, TET2, IDH2, | |
indolent, aggressive, or leukemic | medium-sized). | DNMT3A, CTLA4 ALCL: ALK, NPM1, BCL6, PTPN12, SERPINA1, CEBPB, JAK/STAT pathway ATLL: HBZ HSTCL: STAT5B | ||
Based on site of | Based on nuclear | Hodgkin lymphoma | Clonal rearrangement of | |
involvement: | features: | - CHL | IGH gene by | |
nodal vs extranodal | blastic/blastoid vs | - NLPHL | microdissection of LP & | |
mature | HRS cells | |||
BL
DLBCL
FL
GZL
HGL
IVL
LYG
MCL
MZL
PBL
PEL
SLL
AITL
ALCL
ATLL
ENKT
HSTCL
ITL
MF/SS
PML
PTCL, NOS
SEBVT
SPTCL
T-LBL
Based on identifiable causes: Breast implant-associated; chronic inflammation-associated; EBV-associated;
Helicobacter pylori-associated; HHV-8-associated; HTLV-1-associated; MTX-associated; immunodeficiency-associated
Abbreviations: AITL: Angioimmunoblastic T-cell lymphoma, ALCL: Anaplastic large cell lymphoma, ATLL: Adult T-cell leukemia/ lymphoma, BL: Burkitt lymphoma, B-LBL: B-lymphoblastic lymphoma, CHL: Classic Hodgkin lymphoma, DLBCL: Diffuse large B-cell lymphoma, ENKT: Extranodal NK/T-cell lymphoma, nasal type, FL: Follicular lymphoma, GZL: Gray zone lymphoma (DLBCL vs CHL), HGL: High grade B-cell lymphoma (BL vs DLBCL), HRS: Hodgkin-Reed-Sternberg cells in CHL, HSTCL: Hepatosplenic T-cell lymphoma, IGH: Immunoglobulin heavy chain; ITL: Intestinal T-cell lymphoma, IVL: Intravascular large B-cell lymphoma, LP: LP cells in NLPHL, LPL: Lymphoplasmacytic lymphoma, LYG: Lymphomatoid granulomatosis, MCL: Mantle cell lymphoma, MF/SS: Mycosis fungoides/Sezary syndrome, MTX: Methotrexate; MZL: Marginal zone lymphoma, NLPHL: Nodular lymphocyte predominant Hodgkin lymphoma, PBL: Plasmablastic lymphoma, PEL: Primary effusion lymphoma, PML: Primary mediastinal large B-cell lymphoma, PTCL, NOS: Peripheral T-cell lymphoma, not otherwise specified, SEBVT: Systemic EBV+ T-cell lymphoma of childhood, SLL: Small lymphocytic lymphoma, SPTCL: Subcutaneous panniculitis-like T-cell lymphoma, TCR: T-cell receptor, T-LBL: T-lymphoblastic lymphoma
Cell type and origin (primary site) of metastatic tumors are important because they help guide specific treatment and management of patients. Without any clinical information, pathologists attempt to determine cell type of the metastatic tumor by morphology, histochemistry, immunostaining, and a genetic approach. Usually, it is not difficult to diagnose metastatic tumors in the lymph node because tumor cells are found primarily in lymph node sinuses. The tumor cells tend to form aggregates or sheets as they form tight junctions with nearby tumor cells. Moreover, the morphology of most metastatic tumor cells is different from that of immune cells in the lymph node. However, at times, the metastatic tumor cells can look like lymphoid cells or other accessory cells in the lymph node such as histiocytes, follicular dendritic cells, and interdigitating reticulum cells. Leukemic infiltration, poorly-differentiated neuroendocrine carcinoma (“small cell carcinoma”), metastatic invasive lobular carcinoma, and a number of other metastatic tumors cause difficulties in making a definitive diagnosis because they share morphologic similarities to malignant lymphoma. An experienced pathologist should spend time looking for clues in histologic sections such as immature eosinophils (eosinophilic myelocyte) in leukemic infiltration, nuclear debris along the blood vessel wall in small cell carcinoma, and a large PAS+ cytoplasmic globule by histochemistry in invasive lobular carcinoma.
In case of metastatic carcinoma to the lymph node, common types include adenocarcinoma, squamous cell carcinoma, neuroendocrine carcinoma, urothelial carcinoma, clear cell carcinoma, mucoepidermoid carcinoma, anaplastic carcinoma, and metastatic carcinoma from special types of salivary gland tumors, thyroid gland tumors, pancreatic cancers, gynecologic cancers, etc. In terms of other non-hematologic malignancy, metastatic melanoma, germ cell tumor, and sarcoma are quite common. At present, several types of sarcoma can be diagnosed, even with a core needle biopsy.15 Sarcomas tend to spread via the hematogenous route, however, sarcomas with potential for lymph node metastasis include rhabdomyosarcoma, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, and vascular sarcoma.16 Another important issue is how to determine the nature of the obtained tissue for pathologic examination. Sometimes, it is difficult to distinguish between a lymph node and a soft tissue mass, especially when the subcapsular lymph node sinus cannot be identified. A core needle biopsy is certainly more challenging than an incisional biopsy, especially when the tumor extends into the perinodal soft tissue. In practice, a schwannoma may look like an enlarged lymph node clinically, but surgeons can
identify it as a soft tissue mass during excision. Anyway, a number of schwannoma can be missed and submitted to pathology laboratory as “a lymph node.” Pathologists usually demonstrate that it is schwannoma – not a lymph node. A core needle biopsy of a schwannoma in most published articles is claimed to lead to more accurate diagnosis than fine needle aspiration due to adequate tissue for evaluation.17
Before the advent of immunostaining, histochemistry was used quite frequently. It helps in identifying mucin production in metastatic adenocarcinoma, melanin pigments in metastatic melanoma, or PAS+ intranuclear inclusion in neoplastic plasma cells of lymphoplasmacytic lymphoma, other small B-cell lymphoid neoplasm with plasmacytic differentiation, or even nodal involvement by plasma cell myeloma (multiple myeloma). However, in daily pathology practice at present, a panel of immunostaining is preferred and some pathologists have no experience to use histochemistry for identifying certain materials as mentioned above. Anyway, histochemistry is still worthy in places with limited resources.
A panel of immunostaining can be used in different morphologic settings to determine cell types in a malignancy of the lymph node. The principle is to apply a commercially available antibody specific to an antigen of interest in relation to tumor cells with the hope that, after a panel of antibodies, the immunophenotypic findings can be gathered and interpreted with morphologic correlations to achieve a definitive diagnosis of the type of malignancy. Tables 3 to 7 demonstrate panels of immunostaining proposed for use in various morphologic settings. If the clinical impression and morphology support each other, a pathologist can order a marker specific to the suspected tumor. For example, immunostaining for CD56 (neural cell adhesion molecule or NCAM) can be used in a suspected case of neuroblastoma. However, the expected negative marker, such as vimentin, should be included in the immunostaining panel because CD56 is not specific for neuroblastoma. The author once had a case of intra-abdominal mass in a child who was clinically suspected to have neuroblastoma. The marrow sample showed small blue cells that could be neuroblasts. Only immunostaining for CD56 was performed and tumor cells were positive for CD56 but the serum neuron specific enolase (NSE) level was not elevated that was unusual for metastatic neuroblastoma. So, exploratory laparotomy was performed to remove the tumor mass that was proven to be a sarcoma, probably embryonal rhabdomyosarcoma, supported by positive markers for muscle differentiation, vimentin, and CD56. It has been well documented that CD56 can be positive in a
TABLE 3. Panels of immunostaining proposed for use in undifferentiated neoplasm with large cell morphology (at least 3 times small lymphocyte in size) in the lymph node.5,31
AE1/AE3
CD45 (leukocyte common antigen, LCA, or common leukocyte antigen, CLA)
S-100
Epithelial tumors & epithelioid variant of sarcomas
Lymphoma & some leukemia
Melanoma, LCH, histiocytic sarcoma
Dot positive in neuroendocrine carcinoma (need to view at 40x magnification)
Can be negative in some lymphoma cells but when positive, a few positive tumor cells should be kept for hematologic malignancy
Need both nuclear and cytoplasmic staining for positivity
ALK ALK+ large B-cell lymphoma
CD30 ALCL & lymphocyte-depleted CHL
CD56 Large cell neuroendocrine carcinoma
CD68 Histiocytic tumors & monoblastic sarcoma (leukemic infiltration by monoblasts)
CD138 Plasmablastic lymphoma
EMA Epithelioid variant of sarcoma
MPO Myeloid sarcoma (leukemic infiltration by myeloblasts)
Abbreviations: ALCL: Anaplastic large cell lymphoma, CHL: classic Hodgkin lymphoma, LCH: Langerhans cell histiocytosis
number of normal cells and several kinds of tumors, including embryonal rhabdomyosarcoma.18 Thus, a good immunostaining panel should include not only a positive marker but also a negative one for tumors listed in the differential diagnosis.
Tumor markers do not have ideal specificity so that a complete investigation for primary sites is still needed. For example, NKX3.1 is believed to be a marker of prostatic origin in metastatic tumors, but only one out of 349 non-prostatic tumor tissue tested positive for NKX3.1 and that was a case of invasive lobular carcinoma of the breast.19 Even CD45 (leukocyte common antigen), which is regarded as a highly specific marker in the hematolymphoid neoplasm, there are only seven definitive cases from five reports to date of CD45 expression on non-hematologic malignancy, including one primitive sarcoma (most probably rhabdomyosarcoma), four neuroendocrine carcinomas (including small cell carcinoma), one undifferentiated large cell carcinoma, and one NUT carcinoma; three cases were lymph node metastasis.20-24 Panels of immunostaining as shown in
Tables 3 and 4 provide both positive and negative results that should not have any conflicting immunophenotype. For example, AE1/AE3+ carcinoma cells should not have CD45 expression. When AE1/AE3+ CD45+ tumor cells are detected, a search for any technical error should be performed before acceptance of such an abnormal phenotype (AE1/AE3+ carcinoma with aberrant CD45 expression or CD45+ lymphoma with aberrant AE1/AE3 expression). Technical errors can be the cause of abnormal expression when immunostaining is performed manually, such as wrong slide labeling, applying wrong antibody in immunostaining, contamination of antibody by other antibodies during preparation of primary antibody for use, and interpretation of positive tissue control as the result of the test. All these technical errors can be resolved by using the fully automated immunostainer, except the last one that is caused by the pathologist who looks at positive tissue control placed on the same slide of the tested tissue. To prove that the tumor cells have aberrant expression, the pathologist reviews the histologic section and decides the type of malignancy. For example, if the
TABLE 4. A panel of immunostaining proposed for use in undifferentiated neoplasm with small cell morphology (1-2 times small lymphocyte in size) in the lymph node.4,31
AE1/AE3
CD45 (leukocyte common antigen)
S-100
Epithelial tumor & epithelioid variant of sarcomas
Lymphoma & some leukemia
Melanoma (small cell variant)
Dot positive in small cell (oat cell) carcinoma (need to look at 40x magnification)
Can be negative in some lymphoma cells but when positive, even a few positive tumor cells, should keep work-up for hematologic malignancy
CD33 CD34 CD56
CD99 CD123 CD138 MPO
TdT
Myeloblastic infiltration Leukemic infiltration Neuroblastoma, embryonal rhabdomyosarcoma, BPDCN EWS/PNET
BPDCN
Nodal plasmacytoma Myeloblastic infiltration
Lymphoblastic leukemia/lymphoma
Nodal involvement by PCM
Need both nuclear and cytoplasmic staining for positivity
Abbreviations: BPDCN: Blastic plasmacytoid dendritic cell neoplasm, EWS/PNET: Ewing sarcoma/Primitive neuroectodermal tumor, PCM: plasma cell myeloma (multiple myeloma)
morphology is that of carcinoma, then the tumor cells have aberrant CD45 expression. But if the morphology is that of malignant lymphoma, then the lymphoma cells have aberrant AE1/AE3 expression. If the morphology is not conclusive of any type of malignancy, then more markers are needed to support a diagnosis of malignancy. For example, if the AE1/AE3+ CD45+ tumor cells express CD20, CD10, CD79a, PAX5, MYC, BCL2, and BCL6,
but are negative for EMA, CD3, CD5, and MUM1, then the diagnosis should be DLBCL with germinal center B-cell as the supposed cell of origin with triple protein expression of MYC, BCL2, and BCL6 proteins, and aberrant AE1/AE3 expression. But if the AE1/AE3+ CD45+ tumor cells express CK8/18, TTF-1, CK7, CD56, chromogranin, and synaptophysin but are negative for CK20, p40, p63, CD3, CD20, CD30, CD138, and MUM1,
then the diagnosis should be metastatic carcinoma, possibly primary pulmonary large cell neuroendocrine carcinoma with aberrant CD45 expression.
When there is a malignancy of the lymph node, tissue samples should be handled properly so that pathologists can make a definitive diagnosis. However, with limited resources, general pathologists try to separate reactive conditions from the neoplastic process to provide a possible diagnosis of the type of malignancy found in the lymph node such as malignant lymphoma, metastatic carcinoma, metastatic melanoma, metastatic sarcoma, etc. Afterwards, all the slides, corresponding to tissue block(s), and a corresponding pathology report can be submitted to expert pathologists for further consultation.
A review of the clinical history is paramount in clinical practice. It helps in making a clinical impression of the most likely malignancy of the lymph node. Moreover, it
TABLE 5. Panel of immunostaining commonly used in diagnosis of malignant lymphomas and leukemia (after only CD45 expression in immunostaining panels proposed in Tables 3 or 4).5
CD3
CD2, CD5, CD7 CD4 & CD8
TCR-beta (betaF1) TCR-gamma (GTCR)
EMA
PD1, CXCL13, CD10, BCL6, ICOS-1, HGAL
CD21 & CD23 CD30
ALK
Normal T-cells, T-cell lymphoma, T-LBL, and NK/T-cell lymphoma
Same as CD3
Helper & cytotoxic T-cells
Normal T-cells
Normal T-cells & primary cutaneous gamma-delta T-cell lymphoma Positive up to 85% of ALK+ ALCL TFH
FDC meshwork & hyperplasia Activated/transformed lymphoid cells, HRS cell & ALCL
ALK+ ALCL
Usually membrane staining but cytoplasmic staining in NK/T-cell lymphoma or T lymphoblast
Common T-cell markers; aberrant loss of any of them raises concern of neoplastic nature Normal ratio of 2:1 in peripheral lymphoid tissue and blood; double negative (CD4- CD8-) or double positive (CD4+ CD8+) phenotype deems neoplastic
90% of peripheral blood T-cells 10% of peripheral blood T-cells
Epithelial cells, some plasma cells Need at least 2 markers positive for
diagnosis of AITL or nodal peripheral T-cell lymphoma of TFH phenotype
FDC hyperplasia for AITL
Normal activated/transformed lymphoid cells in T-cell zone
CD20 Normal & neoplastic mature B-cells including NLPHL
CD10
Germinal center B-cell (both reactive
& neoplastic), B-LBL
Membranous staining; may be faint positive in SLL/CLL or negative in some B-cell neoplasms; negative in PCM but may be occasional positive
Positive in normal marrow B-cell precursors, normal & neoplastic bile canaliculi (CC), normal & neoplastic renal tubule (RCC); endometrial stromal sarcoma; non-specific
staining in myeloid series in the marrow
CD5 Normal T-cell & neoplastic B-cells in SLL/CLL & MCL
CD23 Normal B-cell, FDC & neoplastic B-cells
in SLL/CLL
Cyclin D1
MCL, PCM, HCL
SOX11
MCL
CD38, CD138
Normal & neoplastic plasma cell
CD5+ CD23+ in SLL/CLL but CD5+ CD23-
cyclin D1+ in MCL; CD21 is an adjunct marker for FDC
Usually negative in small B-cells in normal mantle layer of lymphoid follicle; also positive in epithelial cells in cell cycle Negative in leukemic phase of MCL;
not a sensitive marker in practice
CD38 not equal to CD138 & both not equal to immunoglobulin staining; CD138 also positive in epithelial cell
Nuclear, nuclear + cytoplasmic, or cytoplasmic pattern
TABLE 5. Panel of immunostaining commonly used in diagnosis of malignant lymphomas and leukemia (after only CD45 expression in immunostaining panels proposed in Tables 3 or 4).5 (Continued)
Kappa & lambda Ig light chains
IgG, IgA, IgM
CD3 T-cells
CD15 Neutrophil & HRS cell
CD20
B-cells & NLPHL
CD30
CD45
Negative in HRS cell
Sometimes only paranuclear positivity in HRS cell; less sensitive in formalin-fixed tissue
Negative in typical CHL but variably positive in occasional HRS cells is also accepted Normal activated/transformed lymphoid cells in T-cell zone
Negative in HRS cell
EMA MUM1
PAX5
Activated/transformed lymphoid cells, HRS cell & ALCL
All types of lymphoid cells & up to 50% of ALCL
Epithelial cells, some plasma cells Positive in HRS cell
B lymphoblast to mature B-cell stage;
faint positive in HRS cell
Negative in HRS cell
Normal & neoplastic plasma cells Negative in plasma cell
MUM1 ALK
Evaluation of Ig light chain expression in plasma cells
Evaluation of Ig heavy chain expression in plasma cells
Normal & neoplastic plasma cells, subset of DLBCL
ALK+ large B-cell lymphoma
Normal kappa to lambda ratio of 2:1; when either kappa or lambda more than the other at least 5 times raises the concern of monoclonal plasma cell population (plasmacytic differentiation)
Usually IgG > IgA > IgM; usually performed when kappa+lambda less than estimated number of plasma cells (by morphology or CD38/CD138 immunostaining) in order to determine heavy chain disease
Large B-cell lymphoma with IRF4
rearrangement (need FISH)
CD34
TdT
CD33, MPO CD14, CD68 CD99 CD117 CD123
Hematopoietic stem cells, blasts, endothelial cells
B-LBL, T-LBL
Myeloblast Monocyte/promonocyte/monoblast Immature hematopoietic cell Myeloid & erythroid precursors BPDCN
Less sensitive for blasts than flow cytometry
AML, M0: CD33+ CD34+ but MPO-
Immature morphology
Express on normal early thymocyte Express on normal mast cells Treated as ALL
Abbreviations: AITL: angioimmunoblastic T-cell lymphoma, ALCL: anaplastic large cell lymphoma, ALL: acute lymphoblastic leukemia, AML, M0: Minimally differentiated acute myeloid leukemia, B-LBL: B-lymphoblastic lymphoma/leukemia, BPDCN: blastic plasmacytoid dendritic cell neoplasm, CC: cholangiocarcinoma, FDC: follicular dendritic cell, FISH: fluorescence in situ hybridization, HCL: hairy cell leukemia, HRS: Hodgkin-Reed-Sternberg, Ig: immunoglobulin, MCL: mantle cell lymphoma, NLPHL: nodular lymphocyte predominant Hodgkin lymphoma, PCM: plasma cell myeloma (multiple myeloma), RCC: renal cell carcinoma, SLL/CLL: small lymphocytic lymphoma/ chronic lymphocytic leukemia, TFH: T follicular helper, T-LBL: T-lymphoblastic lymphoma/leukemia
TABLE 6. Panel of immunostaining commonly used in diagnosis of carcinoma and epithelioid variant of sarcoma (after only AE1/AE3 expression in immunostaining panels proposed in Tables 3 or 4)15,31
CK7 & CK20
CDX2 GATA-3
Hepar 1, Glypican-3, Arginase-1
NKX3.1 PAX8 SATB2
CK5/6 +
p40 +
p63 +
Also positive in mesothelioma, BCC, UC Also positive in BCC, UC
p63+ CD45+ AE1/AE3- DLBCL
TTF-1
CK7+/CK20+: Pancreas, bile duct, stomach, urinary bladder
CK20+/CK7-: Colorectum, Merkel cell carcinoma & occasional upper GI
Colorectum Breast
HCC
Prostate, breast
Endometrium, ovary, thyroid, RCC Colorectum
Lung, thyroid
CK7+/CK20-: Breast, endometrium, ovary, lung, thyroid (also positive in malignant mesothelioma)
CK7-/CK20-: Adrenal cortical carcinoma, prostatic carcinoma, HCC, RCC, neuroendocrine carcinoma of lung & GI tract
Also positive in UC, pheochromocytoma, paraganglioma, choriocarcinoma, malignant mesothelioma
Positive in invasive lobular carcinoma PAX8+ CD45+ AE1/AE3- DLBCL
Also positive in neuroendocrine carcinoma, osteosarcoma, BCOR-rearranged sarcoma
CD56 CK8/18
CD34, ERG CD117, DOG1
Desmin, myogenin INI-1 loss
S100 SOX10
TLE-1
Epithelioid Angiosarcoma SDH-deficient GIST Epithelioid RMS
ES, epithelioid MPNST Epithelioid MPNST Clear cell sarcoma
Synovial sarcoma
Loss of SDH subunit B
Also positive in melanoma & LCH Also positive in melanoma
Also positive in ES, ESS, EWS, MPNST,
schwannoma, and SFT
Chromogranin A Synaptophysin Ki-67
+
+
+
+
>20%
Also + in other types of cancer
<20% in neuroendocrine tumor
Abbreviations: BCC: basal cell carcinoma, DLBCL: Diffuse large B-cell lymphoma, ES: epithelioid sarcoma, ESS: endometrial stromal sarcoma, EWS: Ewing sarcoma, HCC: hepatocellular carcinoma, LCH: Langerhans cell histiocytosis, MPNST: malignant peripheral nerve sheath tumor, RCC: renal cell carcinoma, RMS: rhabdomyosarcoma, SCC: squamous cell carcinoma, SDH-deficient GIST: succinate dehydrogenase-deficient gastrointestinal tumor, UC: Urothelial carcinoma
TABLE 7. Panel of immunostaining commonly used in diagnosis of malignant melanoma and sarcoma (after only S100 expression in immunostaining panels proposed in Tables 3 or 4)31
SOX10
HMB45
Melan A
Also positive in clear cell sarcoma & epithelioid MPNST
Also positive in angiomyolipoma
Also positive in adrenal cortical carcinoma & stromal sex cord tumor
See epithelioid variant of sarcoma in Table 6
Abbreviation: MPNST: malignant peripheral nerve sheath tumor
helps to exclude reactive conditions, infectious processes, and abnormal immune reactions, especially drug reactions and immunodeficiency states, as discussed earlier. The location of the enlarged lymph node implies possible causes such as metastatic CA breast in the axillary lymph node in female patients, metastatic nasopharyngeal carcinoma in level II (upper jugular group) of cervical lymph nodes, metastatic CA thyroid in level IVa (lower jugular group), IVb (medial supraclavicular group), or VIb (deeper pre- laryngeal/pre-tracheal group) of cervical lymph nodes, metastatic CA stomach in left supraclavicular lymph node, and metastatic melanoma in inguinal lymph node. However, at times, unexpected metastatic tumors are observed in unusual locations such as the CA prostate with metastasis to the cervical lymph node.25 However, the most difficult case is cancer of unknown primary (CUP) in clinical practice. CUP rarely presents in lymph node only (LNCUP), according to a series from MD Anderson Cancer Center. LNCUP has better clinical outcomes than CUP in general or CUP with predominant bone disease.26 While axillary LNCUP in women is treated as CA breast with axillary lymph node metastasis, histologic type of adenocarcinoma seems to have better clinical outcomes than other histologic types.27 In a LNCUP case with only one lymph node group positive for metastatic carcinoma, it may be possible to track the primary site according to sentinel node theory, but it is limited to one organ with one direction of lymphatic drainage. It cannot be applied to supraclavicular lymph nodes that receive lymphatic drainage from many organs or lymph node metastasis with more than two directions of lymphatic drainage.28
General practitioners, especially ones who perform tissue biopsy, can help by paying attention to the details of the tissue obtained. Most cellular tumors have soft to firm light brown tissue texture with or without accompanying hemorrhaging or necrosis. White tough fibrous tissue should be avoided and if the tissue biopsy looks like that, doing another biopsy to obtain more representative tissues is a must or the pathology report will come back as “fibrotic tissue obtained, please do another biopsy.” In the past, pathologists preferred complete excision of the enlarged lymph node so that a complete evaluation could be performed. Fine needle aspiration (FNA) of the lymph node is not recommended in suspected cases of malignant lymphoma. However, technical advances have provided options for improvements such as a FNA accompanied by flow cytometry for lymphoma panel. However, core needle biopsy is widely accepted in practice but a large panel of immunostaining is required to obtain more information to compensate the limited histologic evaluation.
There is no doubt that effective communication between pathologists and physicians is the best way to achieve a definitive diagnosis of a malignancy based
on relevant laboratory investigations in addition to good clinical history as mentioned earlier. Even better access via a laboratory information system (LIS) allows pathologists to find relevant laboratory results easier, while a discussion of the case with attending physicians usually reveals important issues of concern about the diagnosis of tumor type. However, in a small number of cases, it is very difficult to acquire a definitive definite diagnosis when the tumor cells do not differentiate well (undifferentiated tumor).
The way pathologists handle cell or tissue samples by conventional morphologic evaluation and panels of immunostaining provides insight to general practitioners to understand the diagnostic process in pathology. In terms of quality assurance, laboratory work involves pre-analytic, analytic, and post-analytic phases. The pre-analytic phase focuses on specimen collection. In this step, the general practitioner needs to know how to obtain the specimen correctly and choose the appropriate test. Regarding pathologic diagnosis, FNA, core needle biopsy, incisional biopsy or excision of the enlarged lymph node should be selected based on clinical information as well as accessibility to the lesion of concern. FNA of the cervical lymph node with a clinical concern of nasopharyngeal carcinoma or cancer of the head and neck region is deemed appropriate. In contrast, this process is not rewarding for malignant lymphoma unless a flow cytometry of fresh samples from FNA is performed at the same time. So, FNA is not recommended in a suspected case of malignant lymphoma where flow cytometry is not available. Instead, excision of the enlarged lymph node is recommended. In places with limited resources, imprints of a fresh cut surface of the lymph node provide cytologic features of lymphoma cells. A good cytotechnologist or even a well-trained hematologist can make a definitive diagnosis from well prepared lymph node imprints, such as Burkitt lymphoma.
Another important issue in the pre-analytics phase is the quality of tissue sample. It should be handled properly so that the slides stained with hematoxylin and eosin (H&E) can be examined by pathologists without interference in the interpretation process. The tissue sample should be fixed in a good volume of neutral buffered formalin (10 times the volume of the tissue sample) for at least two to three hours prior to tissue processing in the laboratory. If core needle biopsy of the lymph node is performed, practitioners must make sure
that a proper lymph node tissue is obtained. The tissue should be soft to firm and light brown. If the tissue is tough white fibrous or pale yellow necrotic, more tissue cores are needed. In case of necrotic tissue, microbiologic studies should be considered as well.
The analytics phase depends on a pathologist’s performance. Good quality H&E-stained slides should allow pathologists to gather relevant microscopic findings to make a diagnosis. Recognition of particular patterns should lead to a list of differential diagnosis. Ultimately, it depends on a pathologist’s knowledge of histology, pathology, training background, technical skills, perception, and memory.29 Then, all clinical and pathological findings are analyzed using the pathologist’s knowledge and experience to provide an interpretation of the lymph node biopsy. The diagnosis could be a straightforward textbook case for any malignancy of the lymph node, however, in a problematic case, additional clinical and laboratory information as well as clinical impression must be considered. Special stains, histochemistry, and immunostaining are requested in order to gather more information to decide the nature of tumor cells. At this point, authorization and expenses can take time, depending on the health care system. If the chain of analysis is allowed to flow freely, an experienced pathologist may handle the case efficiently and provide a diagnosis within 24 or 48 hours, based on histologic evaluation, immunostaining, and/or a number of in situ hybridization (ISH) techniques such as ISH for EBV-encoded small RNA (EBER ISH). In some institutes, a double sign-out system will ask two pathologists to look at the case of malignancy diagnosed for the first time in order to confirm a diagnosis before releasing the pathology report.30
The post-analytic phase depends on the attending physician determining whether the pathologic diagnosis of any type of the malignancy of the lymph node is clinically relevant. In some institutes, before releasing the pathology report, the pathologist and the physician who submits the tissue sample have already discussed the case. Any physician who later gets involved in patient management has a right to challenge the diagnosis when it is not relevant to the clinical information. For example, the pathologist gives a diagnosis of CHL but there are a number of clinical findings suggest NHL. The pathologist should be notified and asked to review the case. Moreover, in any malignancy diagnosed from other hospitals, all pathologic materials (slides, corresponding tissue blocks, and pathology report) should be reviewed by an experienced pathologist to confirm the diagnosis before starting any specific treatment.
CONCLUSION
By understanding the basic principles and the types of malignancy found in the lymph node, a careful study of the clinical history, adequate cell or tissue samples from the lymph node, good clinical collaboration with effective communication between pathologists and physicians, and mastery in conventional morphologic evaluation along with an appropriate panel of immunostaining, general practitioners and pathologists could make the diagnostic process easier in order to diagnose the malignancy of the lymph node in most cases.
ACKNOWLEDGMENTS
The author is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.
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