Application of imaging mass spectrometry for the molecular diagnosis of human breast tumors

Abstract

Distinguishing breast invasive ductal carcinoma (IDC) and breast ductal carcinoma in situ (DCIS) is a key step in breast surgery, especially to determine whether DCIS is associated with tumor cell micro-invasion. However, there is currently no reliable method to obtain molecular information for breast tumor analysis during surgery. Here, we present a novel air flow-assisted ionization (AFAI) mass spectrometry imaging method that can be used in ambient environments to differentiate breast cancer by analyzing lipids. In this study, we demonstrate that various subtypes and histological grades of IDC and DCIS can be discriminated using AFAI-MSI: phospholipids were more abundant in IDC than in DCIS, whereas fatty acids were more abundant in DCIS than in IDC. The classification of specimens in the subtype and grade validation sets showed 100% and 78.6% agreement with the histopathological diagnosis, respectively. Our work shows the rapid classification of breast cancer utilizing AFAI-MSI. This work suggests that this method could be developed to provide surgeons with nearly real-time information to guide surgical resections.

Figure 1

AFAI-MSI of samples of (A,C) low-grade breast ductal carcinoma in situ and (B,D) high-grade breast invasive ductal carcinoma. Positive ion mode AFAI-MSI ion images of sample N₄₉ and sample N₂₂ showing the distribution of (A1, B1) m/z 706.5, PC(30:0); (A2, B2) m/z 718.6, PC(32:0) or PE(35:0); (A3, B3) m/z 724.5, PE(34:1); and (A4, B4) m/z 730.5, PC(32:2). Negative ion mode AFAI-MSI ion images of sample N₄₉ and sample N₂₂ showing the distribution of (C1, D1) m/z 295.2, FA(18:2); (C2, D2) m/z 311.2, octadecanoids; (C3, D3) m/z 327.2, FA(22:6); and (C4, D4) m/z 329.2, FA(22:5). Lower magnification images with expanded views of an adjacent H&E-stained section are shown in (E).

Figure 2. AFAI-MSI of (A1) low-, (B1) intermediate-, and (C1) high-grade breast invasive ductal carcinoma in samples N₅, N₆ and N₂₃.

Positive ion mode AFAI-MS ion images of (A1, B1, C1) m/z 782.6, [PC(34:1)+Na]⁺; (A2, B2, B3) m/z 808.6, [PC(36:2)+Na]⁺; (A3, B3, C3) m/z 810.6, [PC(36:1)+Na]⁺; and (A4, B4, C4) m/z 813.6, SM(42:2). Lower magnification images with expanded views of an adjacent H&E-stained section are shown in (D).

Figure 3. AFAI-MSI of breast cancer samples of (A1) low-, (B1) intermediate-, and (C1) high-grade breast ductal carcinoma in situ in samples N₄₉, N₃₉ and N₂₉.

Negative ion mode AFAI-MS ion images of (A1, B1, C1) m/z 295.2, FA(18:2); (A2, B2, C2) m/z 311.2, octadecanoids; (A3, B3, C3) m/z 327.2, FA(22:6); and (A4, B4, C4) m/z 329.2, FA(22:5). Lower magnification images with expanded views of an adjacent H&E-stained section are shown in (D).

Figure 4

The statistical box plots show the ion intensity of (A, B) IDC and DCIS; (C) low-, intermediate-, and high-grade IDC; (D) low-, intermediate-, and high-grade DCIS.

Figure 5. Tumor heterogeneity was assessed based on AFAI-MSI ion images of sample N₄₈.

Positive and negative ion images of (A) m/z 281.2, oleic acid; (B) m/z 295.2, FA(18:2); (C) m/z 329.2, FA(22:5); (D) m/z 703.6, SM(34:1); (E) m/z 718.6, PC(32:0) or PE(35:0); and (F) m/z 734.6, PC(32:0). An optical image of an adjacent H&E-stained section is shown in (G), with the region of breast invasive ductal cancer delineated with a dotted blue line, the region of breast ductal carcinoma in situ delineated by a dotted red line and a magnified border between these two regions. (H) plots the total abundance of the ions m/z 295.24, m/z 703.59, and m/z 720.54 by the distance (mm) along the dotted black line marked.

Figure 6. AFAI-MSI of breast ductal carcinoma in situ with focal micro-invasion.

Positive and negative ion mode AFAI-MS ion images of sample N₂₉ showing the distribution of (A) m/z 281.2, oleic acid; and (B) m/z 706.5, PC(30:0). An optical image of an adjacent H&E-stained section is shown in (C).