ALN samples, 343, from 93 breast cancer patients were included in this study. Patient characteristics are presented in Table 1. They were derived from complete axillary dissection which had been performed due to a previously positive SN or a clinically positive lymph node. The specimens were collected at the two participating German institutes in this study, University Clinic of Schleswig-Holstein in Kiel, and the Albertinen Hospital in Hamburg, Germany. The 93 patients were fully informed about the study and had given written consent before surgery in compliance with the regulations of the local ethical committees of the University of Kiel and Albertinen Hospital.
The 343 lymph node samples were longitudinally cut into four nearly equal slices (a, b, c, d) with a special cutting tool consisting of three blades being either 1 or 2 mm apart, as depicted elsewhere . ALN were categorized into groups according to their size: ALN with a minor axis smaller than 0.4 cm were excluded from the study; lymph nodes with a minor axis between 0.4 and 0.6 cm (group 1) were centrally cut into four slices with the 1–mm cutting tool; ALN between 0.6 and 1.0 cm (group 2) were centrally cut into four slices with a 2-mm cutting tool. Lymph nodes with a minor axis larger than 1.0 cm (group 3) were either halved or cut into several pieces, and each piece, depending on its size, was treated in a similar fashion as described for groups 1 and 2. Alternate slices were allocated to the OSNA method (a&c) and to histological work-up (b&d) at five levels. The slices used for OSNA (a&c) were shock frozen in liquid nitrogen and stored at −80°C before the analysis. Histological analysis was performed for slices b&d as outlined in a different section. Concordance and sensitivity were determined based on the comparison of these two methods in 343 ALN samples. All histological investigations were done without knowing the results of the OSNA method and vice versa. The first 120 histologically negative out of the 343 lymph node samples, as determined by five-level histology, were cut into further levels until no remnants remained. Specificity was calculated based on the extended investigation of these 120 samples in order to avoid sampling errors caused by uninvestigated material.
If discordant results between the OSNA assay and five-level histological examination occurred, the histological work-up of these cases was also extended until no tissue remained in the paraffin blocks. In addition, the homogenates of these discordant cases were also analysed by Western Blot and quantitative RT-PCR (QRT-PCR) as depicted in a different section. Provided that these supplemental analyses gave the same result as the OSNA assay, these samples were excluded from the study cohort because an uneven distribution of the metastases within pieces a, b, c, and d (tissue allocation bias) was likely to be the case.
One-step nucleic acid amplification
OSNA with CK19 mRNA as the marker was previously described in-depth . In short, the lymph node slices a&c were homogenized together in 4 ml of homogenizing buffer Lynorhag, pH 3.5, (Sysmex, Kobe, Japan) on ice. Twenty microliters of this homogenate were further used for automated amplification of CK19 mRNA via reverse transcription loop-mediated isothermal amplification (RT-LAMP) . Real-time amplification was accomplished with the Lynoamp Kit (Sysmex, Kobe, Japan) on the RD-100i (Sysmex, Kobe, Japan). Four lymph nodes can be analyzed in one run. The degree of amplification was detected via a by-product of the reaction, pyrophosphate . The resulting change in turbidity, upon precipitation of magnesium pyrophosphate, was in turn correlated to CK19 mRNA copy number/μL of the original lysate via a standard curve which was established beforehand with three calibrators containing different CK19 mRNA copy numbers. Since no isolation or purification of RNA was required for OSNA, results were available after a total of 30–40 min. The lymph node lysates were stored at −80°C until further use.
If the CK19 mRNA copy number/μL lysate was less than 250 copies/μL, the result was regarded as (−); copy numbers between 250 and 5,000/μL were regarded as (+), and copy numbers larger than 5,000/μL as (++).
Lymph node slices b&d were fixed with neutral buffered formaldehyde and embedded in the same paraffin block. Each slice was identified by color coding.
Two initial H&E sections (representing frozen sections of SN), one initial level, and four additional levels with a 0.1-mm skip space were cut from the 343 blocks. Each level consisted of four 4 μm sections: one was used for H&E staining, one for immunohistochemistry (IHC) with the pan anticytokeratin antibody LU5 (T-1302, Dianova, Germany), one for CK19 IHC (M0888, clone RCK 108, DAKO, Germany), and one spare section. For the specificity study, the paraffin blocks of 120 histologically negative samples, as judged by five-level histological work-up, were cut into further levels until no remnants remained. IHC was performed according to a standard protocol. Shortly, deparaffinised sections were cooked in a pressure cooker in Tris–ethylenediaminetetraacetic acid–sodium citrate buffer, pH 7.8, for 4 min. After blocking, incubation with the primary antibody was performed for 40 min and with the secondary antibody for 30 min. Visualization was done with diaminobenzidine tetrahydrochloride (Vector, Burlingame, CA, USA). Staining with the LU5 antibody was done using the NEXES staining automat and the I-View-Kit (Ventana, Illkirch, France).
Metastatic deposits were recorded, according to the TNM classification of UICC 6th and AJCC 6th edition [25, 26] as isolated tumor cells (ITC) if their largest diameter was smaller than 0.2 mm, as micrometastases if they were larger than 0.2 mm but not larger than 2 mm in diameter, and as macrometastases if they were larger than 2 mm in diameter. In concordance with the TNM designation of ITC as pN0(i+), lymph node samples were only regarded as positive if at least one micrometastasis or macrometastasis was found. Consequently, lymph nodes with ITC were considered as negative in this study.
Western Blot as part of discordant case investigation
Twenty microliters of the homogenate of discordant specimens were analyzed by Western Blot for CK19. The procedure was recently described in detail . In short, the lysate was mixed with 10 μL loading buffer containing 150 mM Tris–HCl, 300 mM dithiothreitol, 6% sodium dodecyl sulfate (SDS), 0.3% bromophenol blue, and 30% glycerol. The solution was boiled and subjected to electrophoresis on a 7% (w/v) polyacrylamide gel in the presence of SDS (PAG Mini; Daiichi Pure Chemicals, Tokyo, Japan). Proteins were transferred to an Immobilon-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Billierica, MA, USA). After the blocking procedure, the blot was incubated with the primary anticytokeratin 19 antibody A53-B/A2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed with the ECL-Advance detection kit (GE Healthcare, Chalfont St. Giles, UK). CK19 protein concentrations of the lysate, as expressed in nanograms per microliter of the original lysate, were determined on the basis of a previously performed standard curve created with four calibrators of known CK19 protein concentration (Biodesign, Saco, ME). The cutoff value for CK19 protein expression of 0.13 ng/μL (mean + 3 standard deviations = SD) was assigned as previously presented .
QRT-PCR as part of DCI
Total RNA was extracted with the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) from 200 μL of each discordant sample lysate. The quality of the isolated RNA was controlled by performing QRT-PCR of the housekeeping gene beta-actin. QRT-PCR was carried out with CK19 and two breast cancer-specific markers, SPDEF (SAM pointed domain containing ETS transcription factor), and FOXA1 (forkhead box A1).
QRT-PCR was performed on the ABI Prism 7700 detector in duplicates (Applied Biosystems, Foster City, CA, USA). Two microliters of RNA was subjected to one-step QRT-PCR with QuantiTest SYBR Green (QIAGEN, Hilden, Germany) as described by the manufacturer. Primer sequences for CK19, SPDEF, FOXA1, and beta-actin amplification are listed elsewhere [20, 21].
The cutoff levels for each marker as expressed in threshold cycles (ct) were assigned in reference to data obtained with several histologically negative and positive lymph nodes, respectively. The cutoff levels were as follows: 24 for beta-actin, 31.5 for CK19, 33.8 for FOXA1, and 31.6 for SPDEF.