Journal of Surgical Oncology 2009;99:260–264

REVIEW

Lymph Node Assessment: Issues in Pathology

MAHMOUD A. KHALIFA, MD, PhD, FRCPC1* AND ANDY SMITH, MD, FRCSC

2

1Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada2Department of Surgical Oncology, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada

Metastasis to regional lymph nodes detected in cancer surgery specimens is a significant prognosticator and is also critical for subsequent planning

of therapeutic options. Consequently, the status of regional lymph nodes has become a mandatory element of the synotic reporting on cancer

resection specimens. Although pathologic reporting of lymph node metastasis is conceptually straightforward, several subtle processing and

reporting issues deserve discussion. This review addresses pathology specific issues with particular emphasis on non-breast, non-cutaneous

cancers.

J. Surg. Oncol. 2009;99:260–264. � 2008 Wiley-Liss, Inc.

KEY WORDS: node assessment; lymph node pathology; nodal metastasis; cancer staging; quality reporting

INTRODUCTION

Lymph node assessment becomes an issue for the pathologist from

the moment the surgeon transfers the specimen from the operating

room to the pathology suite. This transfer is completed with varying

degrees of detail being supplied to indicate the clinical scenario [1]. At

this point the pathology team begins the process of specimen handling,

gross evaluation and microscopic assessment.

PROCESSING ISSUES WITH GROSSINGAND HANDLING

Lymph nodes can be dissected out of the fresh specimen or after its

fixation in formalin. Since lymph nodes are firmer than the surrounding

fat, they can be identified by diligent, gentle squashing of fat with one’s

fingers. Bouin’s solution has traditionally been regarded as a good

fixative that could enhance detection. Other special fixatives have been

used as revealing solutions to help identify smaller lymph nodes [2–4]

but with varying success in different specimen types [5]. The decision

as to whether to use visually enhancing revealing solutions on routine

basis and whether the search for lymph nodes should be done in the

fresh versus fixed specimen remain user-dependent, with no uniform

guidelines across different pathology laboratories or different speci-

men types. In general, these decisions are made on case-by-case basis

in accordance with the local standard operating procedures of each

laboratory.

Lymph nodes identified by gross assessment are counted with the

understanding that the most accurate and final count will be entered

into the pathology report after microscopic examination. Lymph nodes

are measured either individually or collectively with recording of the

sizes of the smallest and the largest nodes. Each detected lymph node,

depending on its size, is then serial sectioned perpendicular to its long

axis at 0.3 cm intervals. Some prosectors may choose to section lymph

nodes through the hilum in order to increase the chances for detecting

small metastases in this area. Slicing lymph nodes into thin sections is

practically more important than the exact plane of sectioning [6]. A

representative section of a grossly involved lymph node is considered

adequate. However, it has been recommended that all sections of

grossly negative lymph nodes be submitted for further processing and

microscopic examination [7,8]. It is advisable to ensure that all slices

of the lymph node(s) submitted in the same cassette be of comparable

thickness and size. Significantly different slice thickness within the

same cassette may lead to loss of tissue, and potentially micro-

metastasis, from the thinner slices while the histotechnologist trims the

block in an attempt to obtain a complete section of the thicker slice.

In the gross description portion of the pathology report it is

important to document the number of lymph nodes submitted

for sectioning and the specifics of their submission in each cassette.

Without this clear and detailed documentation, the exact number of

nodes can only be speculated leading to potentially serious interpreta-

tion problems (Fig. 1). Even with clear documentation of the

methodology used, confusion may still arise in rare situations. One

example is when a prosector identifies three lymph nodes; each

measuring 0.5 cm in greatest dimension, bisects each node and submits

all resultant six slices in the same cassette. If the microscopic

examination detects metastases in two of the slices on the final section,

the pathologist will not be able to conclusively decide whether the

metastatic lesions were actually present in one or two of the three

nodes originally submitted. The difference between metastasis to one

versus two lymph nodes could be important, depending on the clinical

setting. It is therefore, advisable that only slices of the same lymph

node be submitted together in the same cassette, unless one is dealing

with multiple small (less than 0.3 cm) nodes that can be submitted

together without bisecting or cutting.

*Correspondence to: Mahmoud A. Khalifa, MD, PhD, FRCPC, LaboratoryMedicine and Pathobiology, University of Toronto, Sunnybrook HealthSciences Centre, 2075 Bayview Avenue, Room E-400, Toronto, Ontario,Canada M4N 3M5. Fax: 416 480-4271.E-mail: mahmoud.khalifa@sunnybrook.ca

Received 6 October 2008; Accepted 8 October 2008

DOI 10.1002/jso.21198

Published online 2 December 2008 in Wiley InterScience(www.interscience.wiley.com).

� 2008 Wiley-Liss, Inc.

INTERPRETATIONAL ISSUES WITHMICROSCOPY AND REPORTING

For the purpose of microscopic assessment of lymph node

metastases, a lymph node is defined as the lymphoid tissue of any

size with a recognizable internal nodal architecture including lymphoid

follicles, with or without germinal centers and lymphoid sinuses and is

surrounded by a fibrous capsule. Typically, a lymph node is round or

oval with a smooth contour. Irregular structureless collections of

lymphoid tissue with no fibrous capsule located in the fibroadipose

connective tissue around the resected organ do not count as lymph

nodes. When such irregular lymphoid collections, located in the

vicinity of an invasive carcinoma are involved by malignant cells, it is

regarded as part of the original tumor invading its surrounding

fibroadipose connective tissue. This distinction becomes particularly

important when the final count of lymph nodes, positive and/or

negative, will alter the pN categorization of the tumor. On the other

hand, direct extension of the primary tumor into a lymph node is

classified as a lymph node metastasis and is taken in consideration

while assigning a pN category [9].

The term macrometastasis refers to the intranodal presence of a

cluster of metastatic tumor cells larger than 2.0 mm. When metastatic

nodules are 0.2–2.0 mm in greatest dimension, they are classified as

micrometastasis. Similar to a macrometastasis, a micrometastasis

upgrades the pN category but is reported as pN1(mic). In many

diseases sites, the prognosis for patients with a solitary micrometastasis

is believed to be better than those with larger metastatic deposits.

However, the significance of multiple micrometastases in one lymph

node or multiple lymph nodes with micrometastases remains uncertain.

Multiple separate foci of micrometastases are still classified as

pN1(mic) [10,11]. The number of nodes that contain micrometastases

should be clearly specified in the pathology report since this may affect

treatment [10]. On the other hand, isolated tumor cells (ITCs), are

defined as single or small clusters of cells smaller than 0.2 mm in

greatest dimension, do not affect the pN categorization of the tumor.

Since ITCs do not usually induce recognizable histologic evidence of

malignant activity such as surrounding desmoplastic reaction, they

could be easily overlooked in H&E-stained routine tissue sections.

Their detection largely depends on the use of ancillary techniques

which are not routinely implemented. In fact, the use of immunohis-

tochemistry is still not recommended in routine examination of lymph

nodes for metastasis. The biologic significance of ITCs is unknown. It

has been recommended in breast pathology to use the pN0(iþ) and

pN0(i�) subcategories to distinguish between tumors in which the

ITCs are detected by the use of immunohistochemistry from those in

which detection is done in routinely stained H&E sections [12].

Similarly, the subcategories of pN0(molþ) and pN0(mol�) were

introduced to distinguish cases in which molecular methods such as

DNA analysis and reverse transcriptase polymerase chain reaction are

used [13]. It is also recommended that reporting of ITCs in colorectal

cancer follows the same principles [11].

It is important to remember that the pN category refers to the

status of regional lymph nodes. Metastases to non-regional lymph

nodes are reported under the pM category [9]. As expected, the

anatomic definition and guiding landmarks of ‘‘regional lymph nodes’’

are organ-dependent. Accordingly, clear communication between

surgeons and pathologists becomes crucial in cases where the exact

site of a submitted lymph node or a group of lymph nodes is ambiguous

and could make the difference between a pN and a pM categorization.

TECHNICAL ISSUES WITH NODE COUNTS

In order to accurately count regional lymph nodes, the pathologist

needs to include both lymph nodes present along the feeding blood

vessels that are usually located few centimeters from the resected

organ as well as the nodes present immediately adjacent to the organ

surface. The former nodes are usually sought for in the surrounding or

separately resected adipose tissue and are submitted in separate

cassettes for processing. Therefore, their sections are examined on

Journal of Surgical Oncology

Fig. 1. A histology slide with six sections of nodal structures whichcould represent 6, 3 (one in toto, one bisected, and one trisected),or just one large lymph node serially sections. Without a cleardocumentation as to how the tissue was submitted, speculative countscould create a serious problem.

Fig. 2. A photomicrograph of the uncinate margin in a pancreatico-duodenectomy specimen depicting a benign superior mesenteric lymphnode located at the surgical margin of excision (H&E 100�).

Process Issues in Pathology 261

separate slides. On the other hand, some organs (e.g., colorectum,

pancreas and lung) usually have lymph nodes imbedded in or

immediately adjacent in their outer layers and therefore, can be

included on the same slide depicting the tumor (Fig. 2). Accordingly, a

tumor nodule in these locations that is separate from the primary

carcinoma, even without histologic evidence of residual lymphoid

tissue is considered as an example of complete replacement of the

lymph node by a metastasis and is classified in the pN1 category as

long as it has the form and smooth contour of a lymph node. On the

other hand, if the nodule has an irregular contour, it should be classified

in the pT category as part of the primary tumor.

Regional lymph nodes are sometimes submitted to the pathology

suite as a separate specimen in a separate container as for example is

the case with prostatic, breast, cervical, uterine, ovarian and head and

neck cancers. Alternatively, regional nodes may be resected en bloc

with the cancer-harboring organ and are present in the same container

as is the case with colorectal, pancreatic, lung, kidney and urinary

bladder cancers. Still, in other cases a combination of both scenarios

may occur. Most pathology laboratories will handle the first scenario

by dissecting the grossly identifiable lymph nodes, submitting them as

previously described and also submit the remainder of the adipose

tissue to ensure that every possible lymph node in the specimen is

examined. This is technically possible because the entire specimen

submitted can usually fit in only few processing cassettes. In the second

scenario however, when less than expected lymph nodes are retrieved,

it is still prudent to go back to the specimen and aggressively submit

more adipose tissue or use a visually enhancing revealing solution.

Complete assessment of the regional lymph nodes ideally entails

removal of a sufficient number of lymph nodes to ensure the assigning

of the highest pN category [9]. Several studies have attempted to

address this issue in tumors of the colorectum [14,15], breast [16,17],

uterus [18], and urinary bladder [19,20]. However, the minimum

number of lymph nodes needed to confidently assign a pN0 category

has not been established in carcinomas of other organs such as ovary,

prostate or pancreas. The final number of resected lymph nodes is

dependent on the extent of the resection (i.e., surgeon factors) as well

as the degree of diligent search for the nodes in the resected specimen

(i.e., pathologist factors). In some cases, fewer than expected lymph

nodes are found even after extensive search. This could be attributed to

the fact that some unusual patients constitutionally have few lymph

nodes or the patient may have gone neoadjuvant chemo or radiation

therapy [6]. In the colorectum, 12 lymph nodes have been suggested as

the minimum number needed to confidently categorize the tumor in the

pN0 category [14]. For breast carcinoma, 16 nodes have been regarded

as a reasonable target to ensure a high level of confidence that the

axillary nodes are negative [17]. A statistical model has been suggested

to provide the clinician with a means for assessing the accuracy of the

number of positive axillary lymph nodes reported in patients with one

to three positive nodes [16]. Although some authors have suggested

that similar guidelines are needed to properly establish the extent of

lymphadenectomy for urinary bladder cancer [19], there has been no

evidence to date to suggest a minimum number of nodes in these

patients [20].

Several standard operating procedures in the anatomic pathology

laboratory are guided by benchmarks with numeric values. Hence the

suggested 12 lymph node benchmark in colorectal cancer cases serves

as a powerful example of a practical tool that can be used, directly or as

a surrogate, to assess many of the technical details involved in the

resection and pathologic assessment of colorectal cancer specimens.

The recommendations for its adoption as a quality indicator have

helped to lever efforts to standardize many procedural details in

anatomic pathology laboratories processing colorectal cancer resection

specimens. Expanding on the use of these benchmarks in order to

correlate them with clinical quality outcomes or to employ them as a

regulatory tool by third party payers is a risky leap in logic [21].

CHALLENGES TO INTERPRETING LYMPHNODE STATUS

Increased focus on the significance of lymph node status has led

to more diligent examination of nodes including bisecting or

serial sectioning large nodes, obtaining deeper sections and spending

more time examining H&E-stained sections under high power

magnification. This increased scrutiny has led to awareness of unusual

situations. Sometimes the pathologist will identify small clusters of

tumor cells that are limited to capsular lymphatics and/or subcapsular

sinus in the absence of nodal parenchymal involvement. It is now

believed that, in the absence of true parenchymal invasion, the mere

presence of tumor cells in subcapsular sinus is still a strong predictor of

overall survival [22,23]. Detection of such subtle tumor cells in

sentinel lymph nodes can predict the presence of more obvious

metastases in non-sentinel nodes [24,25] confirming that these cells

should be regarded as equal to parenchymal metastases found in

routine lymph node assessment.

Another challenge relates to lymph node assessment in resected

specimens following neoadjuvant therapy. In some of these specimens,

a lymph node may have a small collection of cell-free mucin, necrotic

debris or cholesterol clefts mixed with hemosiderin-laden macro-

phages. As long as no viable tumor cells are identified, these lymph

nodes are reported as negative (i.e., ypN0). Describing these findings in

a comment may be warranted to document that this finding may signify

the presence of a pre-treatment metastasis that was totally eradicated

by the neoadjuvant therapy.

A further unusual situation arises when a positive lymph node is

identified at the surgical margin, either in its entirety or, less

commonly, with the surgeon’s cut actually going through its

parenchyma. Some of the examples for this scenario include the root

of mesentry in a colectomy specimen, the edge of a mastectomy

specimen with an intramammary lymph node, and a superior

mesenteric lymph node in a pancreatico-duodencetomy specimen.

Since the metastasis in these situations is limited to the nodal

parenchyma it is counted under the pN1 category but the actual

surgical margin is still reported as negative. A description of this

finding is warranted in a comment in order to document its occurrence.

It is also worth mentioning that extranodal soft tissue extension of the

tumor into adjacent fat or muscle has been traditionally regarded as

a poor prognosticator since it is generally associated with a higher

number of positive lymph nodes. It is therefore, recommended to

document the presence of this finding in the pathology report. In some

cases, the extranodal extension is so extensive that individual nodes

cannot be separated and reliably counted. In these cases, the number

of positive lymph nodes may need to be estimated [6]. Whether

extranodal extension could be considered as an indication for

postoperative irradiation is still debatable and is certainly not the case

in every tumor site [26].

INCREASED SCRUTINY OF LYMPH NODES

Twenty-five percent of patients with Stage II colon cancers will

develop recurrence and die from colon cancer. It has been suggested

that further pathological investigations beyond routine histopatho-

logical evaluation such as immunohistochemistry (IHC) and serial

sectioning and/or reverse transcriptase-polymerase chain reaction

assay (RT-PCR), may identify node negative patients at high risk of

recurrence [27]. Studies of increased scrutiny or ultrastaging of lymph

nodes in colon cancer patients have demonstrated that this approach

does identify additional patients with lymph node metastases and this

may impact prognosis. Iddings et al. [27] completed a meta-analysis of

10 articles that assessed the prognostic effect of micro-metastases. IHC

identified a micrometastases in 32% of patients and the 3-year disease

free survival tended to be shorter (76% vs. 80%) but did not reach

Journal of Surgical Oncology

262 Khalifa and Smith

statistical significance. Three studies assessed RT-PCR (total 173

patients) and identified that 64 patients (37%) were upstaged and

overall survival in these patients was significantly worse at 3 years

(78 vs. 97%). The authors postulated that limited nodal sampling might

explain the non-significant survival impact of IHC-detected micro-

metastases versus RT-PCR which is associated with a more complete

nodal analysis and strongly advocated for standardized lymph

node harvesting and processing methodologies and also suggest that

lymphatic mapping that could be used to further identify micro-

metastases [27].

PRACTICAL APPROACHES TO AMPROVINGLYMPH NODE ASSESSMENT

As indicated earlier, in cases where the regional lymph nodes are

submitted to the pathology suite in a separate container, processing

the entire tissue for microscopic examination is a safe and reliable

approach ensuring that all nodes are examined. Due to the relative

small size of these specimens, processing the entire tissue is neither

laborious nor expensive. In cases where the final node count is still

lower than expected despite going back to the specimen and looking

more diligently for nodes with/or without applying revealing solutions,

a comment in the pathology report is usually needed to document the

additional effort spent in this regard.

Processes in the pathology unit influence the number of lymph

nodes examined [28]. In particular, the use of the synoptic format in

order to standardize reporting on all clinically relevant tumor

parameters enhances quality and consistency. The simple act of

adopting a synoptic reporting format of colorectal cancer has been

shown to boost diligence in retrieving lymph nodes and increase their

number [29]. It has also been shown earlier that superior node retrieval

is achieved in hospitals where pathologists’ assistants worked

suggesting that they might have better performance than pathologists

and residents [30,31].

Finally, it seems likely that optimal assessment of lymph nodes and

integration of the information into the overall clinical management of

patients occurs where efforts are taken to improve processes in the

pathology suite and also to optimize communication between

clinicians and administrators. Although barriers that are potentially

amenable to quality improvement include technical issues and need for

pathology assistants efforts should also be directed at improving

organizational support for multidisciplinary interaction (communica-

tion between surgeons and pathologists) and quality improvement

capacity (change leaders or capacity for monitoring) [1].

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