Detection of Metastatic Breast and Thyroid Cancer in Lymph Nodes by Desorption Electrospray Ionization Mass Spectrometry Imaging
- 1.4k Downloads
Ambient ionization mass spectrometry has been widely applied to image lipids and metabolites in primary cancer tissues with the purpose of detecting and understanding metabolic changes associated with cancer development and progression. Here, we report the use of desorption electrospray ionization mass spectrometry (DESI-MS) to image metastatic breast and thyroid cancer in human lymph node tissues. Our results show clear alterations in lipid and metabolite distributions detected in the mass spectra profiles from 42 samples of metastatic thyroid tumors, metastatic breast tumors, and normal lymph node tissues. 2D DESI-MS ion images of selected molecular species allowed discrimination and visualization of specific histologic features within tissue sections, including regions of metastatic cancer, adjacent normal lymph node, and fibrosis or adipose tissues, which strongly correlated with pathologic findings. In thyroid cancer metastasis, increased relative abundances of ceramides and glycerophosphoinisitols were observed. In breast cancer metastasis, increased relative abundances of various fatty acids and specific glycerophospholipids were seen. Trends in the alterations in fatty acyl chain composition of lipid species were also observed through detailed mass spectra evaluation and chemical identification of molecular species. The results obtained demonstrate DESI-MSI as a potential clinical tool for the detection of breast and thyroid cancer metastasis in lymph nodes, although further validation is needed.
KeywordsMass spectrometry imaging Metastatic cancer Cancer staging Lipid profiles Ambient mass spectrometry
Mass spectrometry imaging (MSI) has emerged as a powerful technology for biomedical applications, as it allows chemical characterization and visualization of the spatial distribution of molecules in a variety of biological tissues . In particular, cancer tissue diagnosis using ambient ionization MSI approaches has been explored by several research groups . For instance, DESI-MSI, the most commonly used ambient ionization MS technique, has been employed to analyze many primary human cancer tissues, including brain , prostate , gastric , kidney , and breast [7, 8]. Other ambient ionization MSI techniques such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) , rapid evaporative ionization mass spectrometry (REIMS) , and nano-DESI  have been used to investigate the molecular signatures of biological samples and for cancer diagnosis [2, 12, 13]. Notably, ambient ionization MS techniques bring the advantages of speed and simplicity for molecular evaluation of clinical specimens, and features that are highly attractive for real time diagnosis of tissue specimens in clinical practice.
Cancer staging routinely involves evaluation of regional lymph nodes for presence of metastatic cancer cells. The TNM system is widely used for cancer staging, in which “N” refers to the status of nearby lymph nodes to which the primary tumor has metastasized. The nodal status (value of “N”) plays an important role in determining cancer stage and subsequent treatments. Intraoperative lymph node analysis is commonly performed by histopathologic evaluation of frozen sections or touch imprint cytology. In breast cancer surgery, for example, sentinel lymph node dissection has become a standard technique for staging the axilla and determining the extent of surgical resection. In a study performed on 13,062 breast cancer patients, intraoperative frozen section analysis by histopathologic evaluation allowed metastatic breast cancer detection with a mean sensitivity and specificity of 73% and 100%, respectively . Besides traditional histopathology, other molecular and optical imaging techniques have been explored to provide more accurate detection and staging of cancer in lymph node tissues, including one-step nucleic acid amplification (OSNA) , radio-lympho-scintigraphy , Raman spectrometry , and positron emission tomography (PET) [18, 19]. Matrix assisted laser desorption ionization MSI has been successfully used to identify lymph node metastasis in colorectal, colon, prostate, and breast cancers based on protein detection [20, 21, 22, 23]. More recently, DESI-MSI was used to identify lymph node metastasis from esophageal adenocarcinoma and gastric cancer. In gastric cancer, metabolic profile of the metastatic cancer within lymph nodes was found to be similar to that of the primary tumor site when analyzed by DESI-MSI . In esophageal adenocarcinoma, DESI-MSI allowed objective identification of lymph node metastasis based on its primary tumor lipid profile, with an accuracy of 97.7% in comparison to histopathologic evaluation .
Here, we investigated the usefulness of DESI-MSI for detecting and visualizing metastatic breast and thyroid cancers in lymph node samples. Identification of metabolites, fatty acids (FA), glycerophospholipids (GP), sphingolipids (SL), and glycolipids (GL) was performed to molecularly characterize metastatic tissues within lymph nodes and normal lymph nodes. Various subtypes of complex lipids, such as ceramides (Cer), diacylglycerolipids (DG), triacylglycerolipids (TG), glycerophosphoglycerols (PG), glycerophosphoethanolamines (PE), cardiolipins (CL), glycerophosphoserines (PS), and glycerophosphoinositols (PI) were identified. Trends in the changes of molecular patterns, including variations of degrees of unsaturation of lipid acyl chains were observed in the mass spectra profiles obtained. 2D mapping of the molecules within the tissue sections showed that DESI-MSI allows clear discrimination of cancer metastasis from normal lymphatic and supportive tissue, thus proving evidence that DESI-MSI can be used for lymph node staging in breast and thyroid cancer.
Banked frozen human tissue samples, including normal human lymph nodes (n = 18), lymph node with metastatic breast cancer (n = 16), and lymph node with metastatic papillary thyroid cancer (n = 8) were obtained from Cooperative Human Tissue Network (CHTN) under approved IRB protocol. Samples were stored in a –80 °C freezer until sectioned. Tissue samples were sectioned at 16 μm thick sections using a CryoStar NX50 cryostat (Thermo Scientific, San Jose, CA, USA). After sectioning, the glass slides were stored in a –80 °C freezer. Prior to MSI, the glass slides were dried for ~15 min.
A 2D Omni Spray (Prosolia Inc., Indianapolis, IN, USA) was used for tissue imaging with a spatial resolution of 150 μm. DESI-MSI was performed in the negative ion mode from m/z 100 to 1500, using a hybrid LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific). Mass spectra were acquired in centroid mode. The histologically compatible solvent system dimethylformamide:acetonitrile 1:1 was used for analysis.
The same tissue sections analyzed by DESI-MSI were stained using standard H&E staining protocol. Pathologic evaluation was performed using light microscopy by two dedicated surgical pathologists (C.N. and W.Y.).
Lipid species were identified using high mass accuracy measurements and collision induced dissociation (CID) or higher-energy collisional dissociation (HCD) tandem MS analysis. Fragmentation patterns were compared with literature reports in conjunction with data from Lipidmaps database (www.lipidmaps.org) for identification.
2D Imaging Data Analysis
Xcalibur RAW files were converted into images using FireFly data conversion software (Prosolia, Inc. Indianapolis, IN, USA) and then uploaded into the open source imaging software package BioMAP (Novartis).
Results and Discussion
Molecular Characterization of Lymph Nodes
DESI-MSI experiments were conducted to evaluate the distribution of molecular ions in lymph node tissues. Figure 1b displays 13 representative ion images obtained for a normal lymph node tissue sample, showing a homogenous distribution of these molecular ions in normal lymph node. After DESI-MS imaging, the same tissue was H&E-stained and evaluated by histopathology, which confirmed that the tissue was composed of normal lymphocytes. A relatively homogenous distribution of molecular ions was consistently observed for regions with high density of lymphocytes within all the normal lymph node samples analyzed. In some tissues, lymph node capsule tissue, which is composed of dense irregular connective tissue surrounding lymph node cells, was also identified. The capsule tissue presented characteristic mass spectra with high abundance of TG species, detected as Cl adducts in the negative ion mode, when compared with lymphocytes (Supplementary Figure S1).
Imaging of Metastatic Thyroid Cancer in Lymph Nodes
Imaging of Metastatic Breast Cancer in Lymph Nodes
Few ions detected in sentinel lymph node breast cancer metastasis showed inverse distribution patterns compared with the ion image patterns for thyroid metastasis (Figure 2). For example, glutathione (m/z 306.073), which was almost exclusively detected in cancer region of lymph node thyroid metastasis, displayed higher abundance in adjacent lymph node regions instead (marked in green over the optical image of H&E-stained tissue in Figure 4a). Glutathione is a natural tripeptide that plays important roles in various cellular processes, including antioxidation, cell differentiation, proliferation, and apoptosis. Altered levels of glutathione have been reported in various types of tumors . Interestingly, glutathione metabolism is able to play both protective and pathogenic roles, and have been specifically linked to resistance to chemotherapy . Thus, the changes in relative abundances of glutathione observed by DESI-MSI may be patient-dependent rather disease-specific, and should be further evaluated. Other molecules detected, such as FA (22:4) (m/z 331.264), Cer (d34:1) (m/z 572.481), PS (34:1) (m/z 734.499), and PI (38:4) (m/z 887.564), also presented opposite distribution patterns with that of lymph node thyroid metastasis when comparing metastatic and adjacent lymph node tissues.
We have shown that DESI-MSI can be used to detect and visualize human metastatic thyroid and breast cancer tissues within lymph nodes based on alterations in the abundances of lipids and metabolites. 2D DESI-MS images of specific molecular ions enable clear discrimination of metastatic cancer from other histologic features of the tissue sections, including adjacent normal lymph node tissue and other supportive tissues such as fibrosis and adipose tissues. Through comparison of ion images with pathologic evaluation, changes in relative abundances of specific molecular ions were clearly observed, leading to the inference that some ions could serve as diagnostic markers for detection of thyroid and breast cancer metastasis in lymph node tissues.
Comparison of the mass spectra profiles extracted from regions with a predominant histologic feature in the ion images allowed evaluation of molecular changes associated with metastatic breast, thyroid, and normal lymph node tissues. Distributions of small metabolites, such as glutamine, showed opposite spatial distributions in cancer metastasis and the adjacent normal lymph nodes, respectively. Distribution of glutathione was also distinct in different tissue regions, although inconsistencies across different patients were observed. Several other lipid species, including CL, PI, PS, and Cer, allowed discrimination of metastatic cancer and adjacent normal tissues. Similarly, molecular distributions of FA and GP showed the potential to distinguish between breast cancer metastasis and adjacent normal lymph node tissue. In particular, arachidonic acid, FA(20:4), and docosapentaenoic acid, FA(22:5), showed high relative intensities in the metastatic breast cancer regions compared with adjacent normal lymph node and adipose tissues.
Comparisons of normal lymph node and thyroid tumor tissue spectra showed alterations in fatty acyl unsaturation levels for PS, PG, and PI lipid species in thyroid cancer metastasis. GP species with longer FA carbon chains presented higher levels of unsaturation in papillary carcinoma regions of the tissue compared with normal lymph node regions, whereas shorter carbon chains had reduced levels of unsaturation within the tumor regions. Conversely, while breast cancer metastasis showed differences in unsaturation levels between tumor and normal tissue as well, these tissues exhibited the opposite trend, with longer carbon chains with low levels on unsaturation and short carbon chains with high levels of unsaturation being more abundant in the breast cancer tissue than in the adjacent normal lymph node.
In summary, our results demonstrate the potential of DESI-MSI for lymph node diagnosis to aid in the staging of human thyroid and breast cancers. This is the first example of the use of DESI-MSI for investigation of the metabolic signatures of breast and thyroid cancer metastasis. As ambient ionization MS technologies have been increasingly explored for intraoperative cancer diagnosis, we expect DESI-MSI and other ambient ionization MS techniques to be used for cancer staging through lymph node analysis. Note that while this study demonstrates the use of DESI-MS for tissue section imaging, the time needed for sectioning (~15 min) and MS imaging (~30 min) could limit its use for rapid lymph node evaluation. Thus, we are exploring the use of DESI-MSI for lymph node analysis using alternative sample preparation approaches such as touch imprint biopsies, which are routinely used for rapid intraoperative lymph node analysis by cytopathology . Further, we envision DESI-MS to be used to assess specific tissue regions with ambiguous lymph node diagnosis by histopathology, at a faster operating rate, which may improve throughput and accuracy in cancer staging in clinical practice. Although consistent trends in abundances of specific molecular ions were observed for the 42 tissue samples analyzed, further experiments with a larger sample cohort will be pursued to confirm these findings. Importantly, statistical analysis will also be pursued to determine metabolic markers that significantly characterize metastatic breast and thyroid tissues, and to develop statistical classifiers for rapid tissue diagnosis in order to validate the usefulness of DESI-MSI as an accurate technology for cancer staging.
The authors acknowledge support for this work by the NIH/NCI (grant R00CA190783). Tissue samples were provid-ed by the Cooperative Human Tissue Network, which is funded by the NCI.
- 3.Eberlin, L.S., Norton, I., Orringer, D., Dunn, I.F., Liu, X., Ide, J.L., Jarmusch, A.K., Ligon, K.L., Jolesz, F.A., Golby, A.J., Santagata, S., Agar, N.Y., Cooks, R.G.: Ambient mass spectrometry for the intraoperative molecular diagnosis of human brain tumors. Proc. Natl. Acad. Sci. U. S. A. 110, 1611–1616 (2013)CrossRefGoogle Scholar
- 4.Kerian, K.S., Jarmusch, A.K., Pirro, V., Koch, M.O., Masterson, T.A., Cheng, L., Cooks, R.G.: Differentiation of prostate cancer from normal tissue in radical prostatectomy specimens by desorption electrospray ionization and touch spray ionization mass spectrometry. Analyst 140, 1090–1098 (2015)CrossRefGoogle Scholar
- 5.Eberlin, L.S., Tibshirani, R.J., Zhang, J.L., Longacre, T.A., Berry, G.J., Bingham, D.B., Norton, J.A., Zare, R.N., Poultsides, G.A.: Molecular assessment of surgical-resection margins of gastric cancer by mass-spectrometric imaging. Proc. Natl. Acad. Sci. U. S. A. 111, 2436–2441 (2014)CrossRefGoogle Scholar
- 7.Calligaris, D., Caragacianu, D., Liu, X.H., Norton, I., Thompson, C.J., Richardson, A.L., Golshan, M., Easterling, M.L., Santagata, S., Dillon, D.A., Jolesz, F.A., Agar, N.Y.R.: Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis. Proc. Natl. Acad. Sci. U. S. A. 111, 15184–15189 (2014)CrossRefGoogle Scholar
- 8.Guenther, S., Muirhead, L.J., Speller, A.V., Golf, O., Strittmatter, N., Ramakrishnan, R., Goldin, R.D., Jones, E., Veselkov, K., Nicholson, J., Darzi, A., Takats, Z.: Spatially resolved metabolic phenotyping of breast cancer by desorption electrospray ionization mass spectrometry. Cancer Res. 75, 1828–1837 (2015)CrossRefGoogle Scholar
- 9.Walsh, C.M., Reschke, B.R., Fortney, J., Piktel, D., Razunguzwa, T.T., Powell, M.J., Gibson, L.F.: A novel laser ablation electrospray ionization mass spectrometry (LAESI-MS) platform for biomarker discovery in cancer cells. Cancer Res. 72, 4793–4793 (2012).Google Scholar
- 10.Golf, O., Strittmatter, N., Karancsi, T., Pringle, S.D., Speller, A.V., Mroz, A., Kinross, J.M., Abbassi-Ghadi, N., Jones, E.A., Takats, Z.: Rapid evaporative ionization mass spectrometry imaging platform for direct mapping from bulk tissue and bacterial growth media. Anal. Chem. 87, 2527–2534 (2015)CrossRefGoogle Scholar
- 14.Liu, L.C., Lang, J.E., Lu, Y., Roe, D., Hwang, S.E., Ewing, C.A., Esserman, L.J., Morita, E., Treseler, P., Leong, S.P.: Intraoperative frozen section analysis of sentinel lymph nodes in breast cancer patients: a meta-analysis and single-institution experience. Cancer 117, 250–258 (2011)CrossRefGoogle Scholar
- 15.Tsujimoto, M., Nakabayashi, K., Yoshidome, K., Kaneko, T., Iwase, T., Akiyama, F., Kato, Y., Tsuda, H., Ueda, S., Sato, K., Tamaki, Y., Noguchi, S., Kataoka, T.R., Nakajima, H., Komoike, Y., Inaji, H., Tsugawa, K., Suzuki, K., Nakamura, S., Daitoh, M., Otomo, Y., Matsuura, N.: One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin. Cancer Res. 13, 4807–4816 (2007)CrossRefGoogle Scholar
- 16.Albertini, J.J., Cruse, C.W., Rapaport, D., Wells, K., Ross, M., DeConti, R., Berman, C.G., Jared, K., Messina, J., Lyman, G., Glass, F., Fenske, N., Reintgen, D.S.: Intraoperative radio-lympho-scintigraphy improves sentinel lymph node identification for patients with melanoma. Ann. Surg. 223, 217–224 (1996)CrossRefGoogle Scholar
- 20.Pei, H., Zhu, H., Zeng, S., Li, Y., Yang, H., Shen, L., Chen, J., Zeng, L., Fan, J., Li, X., Gong, Y., Shen, H.: Proteome analysis and tissue microarray for profiling protein markers associated with lymph node metastasis in colorectal cancer. J. Proteome Res. 6, 2495–2501 (2007)CrossRefGoogle Scholar
- 25.Abbassi-Ghadi, N., Golf, O., Kumar, S., Antonowicz, S., McKenzie, J.S., Huang, J., Strittmatter, N., Kudo, H., Jones, E.A., Veselkov, K., Goldin, R., Takats, Z., Hanna, G.B.: Imaging of esophageal lymph node metastases by desorption electrospray ionization mass spectrometry. Cancer Res. 19, 5647–5656 (2016)Google Scholar
- 26.Randolph, G.W., Duh, Q.Y., Heller, K.S., LiVolsi, V.A., Mandel, S.J., Steward, D.L., Tufano, R.P., Tuttle, R.M.: Amercan Thyroid Association Surgical, A: the prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid 22, 1144–1152 (2012)CrossRefGoogle Scholar
- 32.Traverso, N., Ricciarelli, R., Nitti, M., Marengo, B., Furfaro, A.L., Pronzato, M.A., Marinari, U.M., Domenicotti, C.: Role of glutathione in cancer progression and chemoresistance. Oxidative Med. Cellular Longevity. (2013)Google Scholar