Ambient ionization mass spectrometric analysis of human surgical specimens to distinguish renal cell carcinoma from healthy renal tissue

Abstract

Touch spray-mass spectrometry (TS-MS) is an ambient ionization technique (ionization of unprocessed samples in the open air) that may find intraoperative applications in quickly identifying the disease state of cancerous tissues and in defining surgical margins. In this study, TS-MS was performed on fresh kidney tissue (∼1-5 cm(3)), within 1 h of resection, from 21 human subjects afflicted by renal cell carcinoma (RCC). The preliminary diagnostic value of TS-MS data taken from freshly resected tissue was evaluated. Principal component analysis (PCA) of the negative ion mode (m/z 700-1000) data provided the separation between RCC (16 samples) and healthy renal tissue (13 samples). Linear discriminant analysis (LDA) on the PCA-compressed data estimated sensitivity (true positive rate) and specificity (true negative rate) of 98 and 95 %, respectively, based on histopathological evaluation. The results indicate that TS-MS might provide rapid diagnostic information in spite of the complexity of unprocessed kidney tissue and the presence of interferences such as urine and blood. Desorption electrospray ionization-MS imaging (DESI-MSI) in the negative ionization mode was performed on the tissue specimens after TS-MS analysis as a reference method. The DESI imaging experiments provided phospholipid profiles (m/z 700-1000) that also separated RCC and healthy tissue in the PCA space, with PCA-LDA sensitivity and specificity of 100 and 89 %, respectively. The TS and DESI loading plots indicated that different ions contributed most to the separation of RCC from healthy renal tissue (m/z 794 [PC 34:1 + Cl](-) and 844 [PC 38:4 + Cl](-) for TS vs. m/z 788 [PS 36:1 - H](-) and 810 [PS 38:4 - H](-) for DESI), while m/z 885 ([PI 38:4 - H](-)) was important in both TS and DESI. The prospect, remaining hurdles, and future work required for translating TS-MS into a method of intraoperative tissue diagnosis are discussed. Graphical abstract Touch spray-mass spectrometry used for lipid profiling of fresh human renal cell carcinoma. Left) Photograph of the touch spray probe pointed at the MS inlet. Right) Average mass spectra of healthy renal tissue (blue) and RCC (red).

Keywords: Cancer; Desorption electrospray ionization; Mass spectrometry; Multivariate statistics; Surgical tissue analysis; Touch spray ionization.

Fig. 1

Negative ion mode TS-MS results from the kidney surgical cases. A) PCA score plot of the TS-MS data. Each object represents a separate sampling-ionization event from the freshly resected tissue. Healthy renal tissue is blue, RCC is red. A total of 18 human subjects are represented, with 16 RCC and 13 healthy renal tissue specimens, respectively. The average sampling events per tissue is five. B) Corresponding PCA loading plot showing that m/z 792 ([PC 34:2 + Cl]⁻), 794 ([PC 34:1 + Cl]⁻), 844 ([PC 38:4 + Cl]⁻), and 885 ([PI 38:4 - H]⁻) are the major contributors to the separation observed in A. C and D) Average mass spectra of the normal and tumor classes, respectively. The SNV normalized signal intensity is plotted in the average spectra.

Fig. 2

Negative ion mode DESI-MSI results from the surgical kidney specimens. DESI-MSI on cryosectioned tissue was conducted after the TS-MS analysis on the fresh tissue. Samples from 20 human subjects are presented, with 16 RCC and 15 healthy renal tissue specimens, respectively. A) The PCA score plot of the DESI-MSI data. Each object represents a mass spectrum obtained from a ROI of a single tissue specimen. Healthy renal tissue is blue, RCC is red. B) The corresponding loading plot for the score plot in A, showing contributions of m/z 788 ([PS 36:1-H]⁻), 810 ([PS 38:4-H]⁻), and 885 ([PI 38:4 - H]) in the separation observed in the score plot. C & D) The SNV normalized average DESI mass spectra of the healthy renal tissue and RCC classes, respectively.