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J Cancer Metastasis Treat 2020;6:5 I http://dx.doi.org/10.20517/2394-4722.2020.13 Page 29 of 38

crucial to provide appropriate cares for patients. However, no technique is the universal gold standard to
detect accurate HER2 status. In this context, we established a multiple reaction monitoring (MRM) assay
to quantitate HER2 protein that improves upon existing methods in differentiating between each HER2
status in FFPE tissue specimens. We developed a targeted proteomic assay based on multiple reaction
monitoring-mass spectrometry (MRM-MS) and quantified levels of HER2-MRM protein in breast cancer
FFPE tissues.

We analyzed a total of 210 breast cancer FFPE tissue specimens, which were comprised of HER2 0 (n = 30),
HER2 1+ (n = 30), HER2 2+FISH- (n = 61), HER2 2+FISH+ (n = 59), and HER2 3+ (n = 30). We applied
normalization factors that can represent the tumor size to simplify the overall experimental work-flow and
raise the accuracy and precision of the results of HER2 quantification. In this context, the ratio between the
quantification data of HER2 peptides by MRM assay and the normalization factor can be a new factor for
determining HER2 status.

To select the most suitable normalization factor that can differentiate ambiguous IHC results of HER2 (HER2
2+FISH- vs. HER2 2+FISH+), which cannot be distinguished by IHC, area under the receiver operating
curve (AUROC) values were calculated by using each normalized value of the 120 HER2 2+ samples.
To determine whether the data generated by MRM matched with the data obtained by IHC and FISH
scores, the quantitative data of a HER2 peptide normalized by a Junctional adhesion molecule A (JAM1)
peptide with the highest AUROC values were used. The Mann-Whitney U test determined that significant
differences were found in all HER2 and FISH groups, and especially the MRM data can distinguish between
HER2 2+FISH- and HER2 2+FISH+ (P < 0.000), which cannot be differentiated by IHC. In addition, the
MRM data distinguished the HER2 positive group that was expected to benefit from trastuzumab therapy
from the HER2 negative group (P < 0.000).

We developed an experimental work-flow that is simple and clear enough to automate by introducing
normalization factors for accurate HER2 status determination through MRM assay. The MRM assay
that we developed clearly distinguished the equivocal HER2 status that could not be classified by the
conventional method, IHC, as well as the overall HER2 classification. Our developed assay using MRM for
determining HER2 status would provide clinicians with valuable diagnostic information and ensure that all
patients whose breast cancers express HER2 proteins have the opportunity to receive proper treatment.

41. Amino-functionalized nanoparticles promote toxicity in ovarian cancer cells by impinging
on autophagy

1
1,2
1
Alessandra Ferraresi , Christian Seca , Suratchanee Phadngam , Chiara Vidoni , Marco
1
1
Palminteri , Ciro Isidoro 1
1 Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del
Piemonte Orientale, Via Solaroli 17, Novara 28100, Italy.
2 Department of Radiologic Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang
Mai 50200, Thailand.

Background: In the last decades, nanotheranostics has obtained great attention for its potential application
in the biomedical field by combining multimodal imaging along with selective targeting therapy on the
[1]
same nanoplatforms . However, the contributions of metabolic, genetic, or epigenetic features of tumor
cells and tumor microenvironment in the cellular response to the nanoparticles have not been fully
[2]
addressed .