# Versatile Mass Spectrometry-Based Intraoperative Diagnosis of Liver Tumor in a Multiethnic Cohort
## 3 Results  
### PESI-MS Analysis  
On the 224 specimens collected by the Department of Hepatobilary and General Surgery of Humanitas Clinical and Research Center of Milan, 311 PESI-MS analyses were conducted.  
Although the majority of analyses agreed with the pathologist's diagnosis, there were a few false positives (specimens defined as non-tumor by the pathologist but tumor by the instrument) and false negatives (specimens defined as tumor by the pathologist but non-tumor by the instrument). There were 266 analyses whose results were concordant with the pathologist's diagnosis, 36 false positive, and 9 false negative (Figure 1). Considering the total number of analyses done by PESI-MS, the diagnostic concordance rate was 85.5% (\(n=266\)).  
Most of the non-concordant results were between probability values of TCP 40-70%. A set of these samples was selected randomly and re-analyzed. Those with a correct assignment confirmed the classification, while some of the borderline samples that had been misclassified the first time were then concordant with the pathologist's diagnosis. Excluding non-concordant results that agreed with the pathologist on re-analyzing a second piece of specimens, the diagnostic concordance rate rose to 95.1% (Figure 2). On the samples that still resulted in being non-concordant, a follow-up was advisable, in order to check if the error came from the instrument or from the pathologist classification.  
Figure 1: Results of the 311 PESI-MS analyses. Results concordant with the pathologist’s diagnosis are reported in green (\(n=266\)), false positive results in red (\(n=36\)), and false negative results in blue (\(n=9\)). For these results, the diagnostic concordance rate was 85.5%.  
The false positive results were concentrated near the threshold of the HCC judgment score (50% TCP), and the concordance of the Italian specimens with the database created with the Japanese specimens was slightly low. This misclassification was supposed to be due to the higher sensitivity of mass spectrometry toward cancer cells compared to the pathologist's subjective diagnosis. False negatives, on the other side, were all with low TCP, where less pathological tissue was present, and it was more difficult for the pathologist to diagnose it.  
To improve the judgment concordance rate of SVM, a set of 30 new samples from Italian patients were added to the database created with Japanese samples (Figure 3). HCC judgment scores evaluated before and after additional training were compared: before the additional training, the peaks of the frequency distribution of HCC and non HCC specimens were around the threshold (Figure 3a), whereas after training, the peaks of the frequency were better separated (Figure 3b).  
The performance of the database built by combining both Japanese and Italian samples was evaluated by the K-fold cross validation method, dividing the 2529 spectra acquired  
Figure 3: Support Vector Machine score distribution of non-HCC specimens (blue) and HCC specimens (pink). (**a**) Analysis of Italian samples (2529 spectra, 180 specimens) using the SVM database built with Japanese samples; (**b**) Analysis of Italian samples (30 samples as Test datasets) using a database built only with Japanese samples (7786 spectra) and with Italian samples (2529 spectra).  
Figure 2: Results of 224 specimens analyzed by PESI-MS, excluding the first incorrect diagnosis. Results concordant with the pathologist’s diagnosis are reported in green (\(n=213\)), false positive results in red (\(n=9\)), and false negative results are reported in blue (\(n=2\)). For these results, the diagnostic concordance rate was 95.1%.  
from Italian samples into K = 10 data sets (Table 2). The judgment concordance rate was 97.6% for non-HCC specimens and 93.9% for HCC specimens. Considering both HCC and non-HCC samples, the judgment concordance rate was 95.5% (see Table 2)