Quantitative molecular analysis of sentinel lymph node may be predictive of axillary node status in breast cancer classified by molecular subtypes

Simonetta Buglioni, Franco Di Filippo, Irene Terrenato, Beatrice Casini, Enzo Gallo, Ferdinando Marandino, Carlo L Maini, Rossella Pasqualoni, Claudio Botti, Simona Di Filippo, Edoardo Pescarmona, Marcella Mottolese, Simonetta Buglioni, Franco Di Filippo, Irene Terrenato, Beatrice Casini, Enzo Gallo, Ferdinando Marandino, Carlo L Maini, Rossella Pasqualoni, Claudio Botti, Simona Di Filippo, Edoardo Pescarmona, Marcella Mottolese

Abstract

To determine the performance of intraoperative one-step nucleic acid amplification (OSNA) assay in detecting sentinel lymph node metastases compared to postoperative histology taking into account breast cancer molecular classification and to evaluate whether the level of cytokeratin 19 mRNA copy number may be useful in predicting the likelihood of a positive axillary lymph node dissection. OSNA assay was performed in a prospective series of 903 consecutive sentinel lymph nodes from 709 breast cancer patients using 2 alternate slices of each sentinel lymph node. The remaining 2 slices were investigated by histology. Cytokeratin 19 mRNA copy number, which distinguishes negative cases (<250 copies), micrometastases (+, ≥250≤5000 copies) and macrometastases (++, >5000 copies), was compared to axillary lymph node dissection status and to the biological tumor profile. Concordance between OSNA and histopathology was 95%, specificity 95% and sensitivity 93%. Multiple Corresponce Analysis and logistic regression evidenced that positive axillary lymph node dissection was significantly associated with a higher cytokeratin 19 mRNA copy number (>5000; p<0.0001), HER2 subtype (p = 0.007) and lymphovascular invasion (p<0.0001). Conversely, breast cancer patients with cytokeratin 19 mRNA copy number <2000 mostly presented a luminal subtype and a negative axillary lymph node dissection. We confirmed that OSNA assay can provide standardized and reproducible results and that it represents a fast and quantitative tool for intraoperative evaluation of sentinel lymph node. Omission of axillary lymph node dissection could be proposed in patients presenting a sentinel lymph node with a cytokeratin 19 mRNA copy number <2000 and a Luminal tumor phenotype.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Correlation between OSNA assay and…
Figure 1. Correlation between OSNA assay and histology results in the entire series of 903 SLNs. Panel A:
The histograms summarize the percentage of concordant and discordant results on SLNs analyzed by OSNA and histology. Panel B: Representative examples of CK19 immunostaining (immunoperoxidase) of a micrometastasis (a) and a macrometastasis (b) in two different SLNs compared to the matched OSNA curves (a-b) (scale bar = 90 µm). Panel C: The histograms show the distribution of −/negative, +/micrometastatic and ++/macrometastatic SLNs analyzed both by molecular and morphological methods. The percentage of micrometastases by OSNA was 11.9% and by histology 7.4%, whereas the percentage of macrometastases by OSNA was 10.4% and by histology 13.4%. Histology included H&E staining and IHC. OSNA−: <250; OSNA+ : >250≤5000; OSNA++: >5000 CK19 mRNA copies/µl.
Figure 2. Relationship between number of SLNs…
Figure 2. Relationship between number of SLNs tested by OSNA and ALND status.
The percentage of negative ALND was significantly higher in patients with 1 OSNA positive SLN (65.8% vs 34.2%). Conversely, the percentage of positive ALND was significantly higher in patients with 2 or more OSNA positive SLNs (66.7% vs 33.3%) (p = 0.004).
Figure 3. Relationship between SLN status and…
Figure 3. Relationship between SLN status and bio-pathological parameters.
The histograms show that OSNA positive results are significantly correlated with (Panel A) poor differentiated tumor (p = 0.039), (Panel B) high proliferation index (p = 0.028) and (Panel C) presence of lymphovascular invasion (LVI p<0.0001).
Figure 4. MCA of the 179 OSNA…
Figure 4. MCA of the 179 OSNA +/++ BC patients stratified by molecular subtypes and relationship between BC subtypes and ALND status in the subset of 91 OSNA+ BC patients.
Panel A: The MCA graph demonstrates that ALND (+) is located in the quadrant containing the most aggressive phenotypes (T2 tumors, positive LVI, HER2 subtype, OSNA++) in contrast to ALND (−) which is associated to more favourable bio-pathological parameters (T1 tumors, absence of LVI, LA subtype). *LA: Luminal A; LB: Luminal B; HS: HER2 subtype; TN: Triple Negative Panel B : The histograms report the distribution of molecular subtypes in the 72 OSNA+ cases with ALND negative and CK19 mRNA <2000 copies/µl (range 270–1900). Panel C : The histograms report the distribution of molecular subtypes in the 19 OSNA+ cases with ALND positive and CK19 mRNA >2000 copies/µl (range 2100–4600).

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