Validation of Breast Cancer Margins by Tissue Spray Mass Spectrometry

Current methods for the intraoperative determination of breast cancer margins commonly suffer from the insufficient accuracy, specificity and/or low speed of analysis, increasing the time and cost of operation as well the risk of cancer recurrence. The purpose of this study is to develop a method for the rapid and accurate determination of breast cancer margins using direct molecular profiling by mass spectrometry (MS). Direct molecular fingerprinting of tiny pieces of breast tissue (approximately 1 × 1 × 1 mm) is performed using a home-built tissue spray ionization source installed on a Maxis Impact quadrupole time-of-flight mass spectrometer (qTOF MS) (Bruker Daltonics, Hamburg, Germany). Statistical analysis of MS data from 50 samples of both normal and cancer tissue (from 25 patients) was performed using orthogonal projections onto latent structures discriminant analysis (OPLS-DA). Additionally, the results of OPLS classification of new 19 pieces of two tissue samples were compared with the results of histological analysis performed on the same tissues samples. The average time of analysis for one sample was about 5 min. Positive and negative ionization modes are used to provide complementary information and to find out the most informative method for a breast tissue classification. The analysis provides information on 11 lipid classes. OPLS-DA models are created for the classification of normal and cancer tissue based on the various datasets: All mass spectrometric peaks over 300 counts; peaks with a statistically significant difference of intensity determined by the Mann–Whitney U-test (p < 0.05); peaks identified as lipids; both identified and significantly different peaks. The highest values of Q2 have models built on all MS peaks and on significantly different peaks. While such models are useful for classification itself, they are of less value for building explanatory mechanisms of pathophysiology and providing a pathway analysis. Models based on identified peaks are preferable from this point of view. Results obtained by OPLS-DA classification of the tissue spray MS data of a new sample set (n = 19) revealed 100% sensitivity and specificity when compared to histological analysis, the “gold” standard for tissue classification. “All peaks” and “significantly different peaks” datasets in the positive ion mode were ideal for breast cancer tissue classification. Our results indicate the potential of tissue spray mass spectrometry for rapid, accurate and intraoperative diagnostics of breast cancer tissue as a means to reduce surgical intervention

Shotgun Lipidomics for Differential Diagnosis of HPV-Associated Cervix Transformation

A dramatic increase in cervical diseases associated with human papillomaviruses (HPV) in women of reproductive age has been observed over the past decades. An accurate differential diagnosis of the severity of cervical intraepithelial neoplasia and the choice of the optimal treatment requires the search for effective biomarkers with high diagnostic and prognostic value. The objective of this study was to introduce a method for rapid shotgun lipidomics to differentiate stages of HPV-associated cervix epithelium transformation. Tissue samples from 110 HPV-positive women with cervicitis (n = 30), low-grade squamous intraepithelial lesions (LSIL) (n = 30), high-grade squamous intraepithelial lesions (HSIL) (n = 30), and cervical cancers (n = 20) were obtained. The cervical epithelial tissue lipidome at different stages of cervix neoplastic transformation was studied by a shotgun label-free approach. It is based on electrospray ionization mass spectrometry (ESI-MS) data of a tissue extract. Lipidomic data were processed by the orthogonal projections to latent structures discriminant analysis (OPLS-DA) to build statistical models, differentiating stages of cervix transformation. Significant differences in the lipid profile between the lesion and surrounding tissues were revealed in chronic cervicitis, LSIL, HSIL, and cervical cancer. The lipids specific for HPV-induced cervical transformation mainly belong to glycerophospholipids: phosphatidylcholines, and phosphatidylethanolamines. The developed diagnostic OPLS-DA models were based on 23 marker lipids. More than 90% of these marker lipids positively correlated with the degree of cervix transformation. The algorithm was developed for the management of patients with HPV-associated diseases of the cervix, based on the panel of 23 lipids as a result. ESI-MS analysis of a lipid extract by direct injection through a loop, takes about 25 min (including preparation of the lipid extract), which is significantly less than the time required for the HPV test (several hours for hybrid capture and about an hour for PCR). This makes lipid mass spectrometric analysis a promising method for express diagnostics of HPV-associated neoplastic diseases of the cervix