Metastasis detection in sentinel lymph nodes: comparison of a limited widely spaced (NSABP protocol B-32) and a comprehensive narrowly spaced paraffin block sectioning strategy

Abstract

The National Surgical Adjuvant Breast and Bowel Project B-32 trial is examining whether patients with initially negative sentinel lymph nodes (SLNs) who have occult metastases detected on deeper levels and cytokeratin immunohistochemistry stains are at risk for regional or distant metastases. The experimental B-32 protocol was designed to detect metastases larger than 1.0 mm by examining sections approximately 0.5 and 1.0 mm deeper into the paraffin blocks (2 levels; wide spacing). This pilot quality assurance study compares detection rates to a comprehensive protocol designed to detect metastases larger than 0.2 mm (multilevel; narrow spacing). All SLNs were sectioned grossly at close to 2.0 mm and all sections embedded in paraffin blocks. For clinical treatment, a single hematoxylin and eosin section was examined from each block. For 54 cases with 1 to 5 SLNs and all SLNs negative, additional cytokeratin immunohistochemistry sections were evaluated every 0.18 mm through the block until no tissue remained. Twenty of 176 (11.4%) blocks harbored occult metastases; the B-32 protocol detected metastases in 11 blocks (6.3%) and 9 additional blocks (5.1%) with metastases were detected on sections that would not have been evaluated (P=0.002; correlated proportions). Median number of levels examined per block on the comprehensive protocol was 11 (range: 3 to 26); the B-32 protocol was fixed at 2 levels (median 2; range: 1 to 2). Median thickness of node sections in the block was 2.1 mm (range: 0.7 to 4.8 mm) and the modal thickness was 2.3 mm. Although more comprehensive sectioning of SLNs detects additional micrometastases, the data suggest diminishing returns and reduced cost effectiveness for the comprehensive strategy.

Figures

Figure 1. Paraffin block sectioning protocols

All sentinel nodes were either bisected or sectioned to produce gross node sections approximately 2.0 mm thick. All gross sections were embedded in paraffin blocks. A. A single hematoxylin and eosin (H&E) stained section was evaluated from the surface of each paraffin block and the results documented in the pathology report for clinical treatment decisions. This protocol is referred to as the B-32 clinical sectioning strategy and was designed to exclude macrometastases larger than 2.0 mm from the “node negative” group. Note that metastases up to 2.0 mm may be missed. B. One H&E and one cytokeratin immunohistochemial (CK-IHC) stained section were evaluated at two levels - approximately 0.5 mm and 1.0 mm - deeper into the paraffin block (four sections total). This two-level, widely spaced protocol is referred to as the B-32 experimental strategy and was designed to exclude micrometastases larger than 1.0 mm from the “occult metastases negative” group. Note that isolated tumor cell clusters (ITC) and micrometastases up to 1.0 mm may be missed. C. One CK-IHC stained section was evaluated every 0.18 mm completely through the paraffin block. This multilevel, narrowly spaced protocol is referred to as the comprehensive strategy and was designed to exclude micrometastases larger than 0.2 mm from the “occult metastases negative” group. Note that ITCs up to 0.18 mm may be missed. Occult metastases identified on sections at approximately 0.5 mm and 1.0 mm into the paraffin block - the sections evaluated for the B-32 occult metastasis study - were compared to the occult metastases identified on the entire set of CK-IHC stained sections. The dotted line represents the initial section used for clinical treatment; all SLN blocks were required to be negative on this initial section to be included in the pilot study.

Figure 2. Location by level and size of occult metastases detected in sentinel lymph nodes

A comprehensive sectioning strategy evaluating cytokeratin immunohistochemical stains every 0.18 mm entirely through the paraffin block was used. Occult metastasis detection rates for two-level narrow spacing (2N), two-level wide spacing (B-32), and four-level wide spacing (4W) protocols were compared to the reference comprehensive multilevel narrow spacing protocol. The comprehensive protocol is statistically significantly different from the remaining sampling protocols. Note that the smallest p-value and lowest rate of metastasis detection is associated with the 2N protocol sampling the most superficial levels of the paraffin block indicating the two-level narrow spacing protocol is the least effective at detecting occult micrometastases. Gray boxes indicate occult metastases no larger than 0.2 mm (isolated tumor cell clusters; ITCs) and black boxes indicate occult metastases larger than 0.2 mm (micrometastases). Each box contains the size in μm (microns) of the largest occult metastasis identified on the level. Blocks 10B and 47B1 contained contiguous micrometastases present on several levels with non-contiguous ITCs on sections superficial and deep to the micrometastasis.

Figure 3. Comparison of maximum metastasis size detected by a two-level wide spaced and a multilevel narrow spaced sectioning protocol

The wide spaced (B-32) protocol examines only two sections at approximately 0.5 mm and 1.0 mm deeper into the paraffin block. The narrow spaced (comprehensive) protocol examines sections every 0.18 mm through the block producing a median of 11 sections per block. Occult metastases were identified in nine additional blocks by the comprehensive protocol (one micrometastasis (25B); 8 isolated tumor cell clusters). Three micrometastases were misclassified as isolated tumor cell clusters by the B-32 protocol (9C, 9D, 47B1).

Figure 4. Frequency distribution of maximum lymph node section thickness in 176 paraffin tissue blocks

Lymph nodes were bisected or sectioned grossly at approximately 2.0 mm maximum thickness, embedded in paraffin blocks, and microscopic sections mounted on slides at 0.18 mm intervals through the block. Histology technicians were instructed to mount sections until no nodal tissue remained visible in the block. The last level containing any microscopic nodal tissue was recorded; any further sections containing only adipose were not included. In general and when possible, two levels were mounted on each slide for cytokeratin staining to reduce expense. Note the higher frequency of the last level occurring on an even numbered section. For thickness calculation, it was assumed that 0.3 mm of tissue had been removed for initial and subsequent block facing prior to the first experimental cytokeratin stain examined. Calculated median and modal thickness was 2.1 and 2.3 mm, respectively.