METHOD FOR PROVIDING INFORMATION FOR CHOOSING BREAST CANCER TREATMENT METHOD BY USING BREAST CANCER ULTRASONIC IMAGE AND GENE INFORMATION

Abstract

The present invention relates to: a method for providing information for choosing a breast cancer treatment method by using a breast cancer ultrasonic image and gene information; a system for choosing a breast cancer treatment method by using a breast cancer ultrasonic image; and a method for providing information required for predicting the prognosis of a breast cancer patient.

Claims

1. A method for providing information for choosing a breast cancer therapy by using an ultrasound image of breast cancer, the method comprising: a phenotype determination step of determining a phenotype of a tumor by using an ultrasound image; a gene information determination step of determining at least one gene information related to breast cancer by using the phenotype of the tumor; and a therapy determination step of determining an individual breast cancer therapy by correlating the determined gene information with gene information associated with a breast cancer therapy.

2. The method of claim 1, wherein the ultrasound image includes a B-mode ultrasound image, a superb microvascular imaging (SMI) ultrasound image, and a contrast-enhanced ultrasound (CEUS) image.

3. The method of claim 2, wherein the phenotype determined through the B-mode ultrasound image includes size, shape, orientation, margin, and calcifications.

4. The method of claim 3, wherein the size is related to at least one gene selected from the group consisting of MIR941-1, IGLV6-57, HIST1H1B, HIST1H3I, ADH1B, PLIN4, and LUZP6; the shape is related to at least one gene selected from the group consisting of MIR941-1, ZFP36L1, SNHG9, H2AFY2, POTEI, UBE2Q2L, FABP7, IGHV3-43, IGKJ5, and IGKJ2; the orientation is related to at least one gene selected from the group consisting of TFF1, AREG, AGR3, TFF3, LINC00993, IGKV2-28, IGLV1-51, IGHV3-73, IGKV3-20, IGLV2-14, IGKV1-12, IGHV4-61, IGKV3D-15, IGHV1-3, IGHV4-4, IGHV1-18, IGHV4-34, IGHV3-74, CALML5, IGHJ5, and IGKJ2; the margin is related to HLA-C gene; and the calcifications are related to at least one gene selected from the group consisting of CALML3, HIST1H4F, IGHV4OR15-8, CCL19, and IGLV8-61.

5. The method of claim 2, wherein the phenotype determined through the SMI ultrasound image includes vascular index, vessel morphology, and penetrating vessel.

6. The method of claim 5, wherein the vascular index is related to at least one gene selected from the group consisting of IGHJ5, MIR1307, IGLV6-57, HLA-C, HIST2H2BE, CALML3, IGKV6-21, OR5P3, and MIR597; the vessel morphology is related to at least one gene selected from the group consisting of HIST1H4D, TUSC1, FZD8, NMI, IGF1R, UBB, SERHL2, NFIL3, CRIPAK, SNHG20, HBA2, and SNHG12; and the penetrating vessel is related to at least one gene selected from the group consisting of HIST1H4D, CST1, TRBC2, SLC25A2, KRT14, MFAP4, IGKV2-40, NFIL3, POTEE, POTEI, ALDH3B2, CRIPAK, IGHJ2, AREG, and IGKJ5.

7. The method of claim 2, wherein the phenotype determined through the contrast-enhanced ultrasound image includes enhancement order, enhancement margin, internal homogeneity, penetrating vessel, and perfusion defect.

8. The method of claim 7, wherein the enhancement order is related to at least one gene selected from the group consisting of IGKV1D-39, CCL3L3, IGHG4, IGKV1D-12, IGKV3D-11, SNHG12, CPB1, MIR562, and VTRNA2-1; the enhancement margin is related to at least one gene selected from the group consisting of STH, TFF1, STC2, AMY2A, HOXB5, IGKV1D-39, PHLDA2, HIST1H2AJ, TRAV14DV4, HIST1H1A, CXCL10, ISG15, IGHV4-39, IGKV3D-15, HIST2H2BF, HIST1H2BM, IGKV2-28, IGHV3-21, CALML5, IGHV1-18, IGKV2-29, IGHG4, IGHJ4, IGHJ5, and IGKJ2; the internal homogeneity is related to at least one gene selected from the group consisting of IGKJ5, HLA-DQA1, HIST1H1B, and IGHV3-74; the penetrating vessel is related to at least one gene selected from the group consisting of AGR2, HIST1H2BI, IGHV4-4, IGLV3-25, IGKV1D-39, IGHV1-2, IGHV3-15, IGKV1-27, IGLV3-1, IGKV2-40, IGKV2D-40, IGHV1-18, HIST1H2AG, IGHV3-33, IGKV1-12, IGKV1-17, IGHG1, TRBV5-6, IGHG4, IGHV4-61, IGKV2-28, IGHV1-8, IGHV4-39, IGHV3-21, IGHV3-9, IGKV3D-15, MIR562, IGHV1-69, IGHV4-31, IGHV1-3, and OR2J3; and the perfusion defect is related to at least one gene selected from the group consisting of HLA-DQA1, AREG, SNHG29, and IGHV3-74.

9. A system for choosing a breast cancer therapy by using an ultrasound image of breast cancer, the system comprising: a database allowing gene information associated with a breast cancer therapy to be retrieved or extracted; a communication unit accessible to the database; a first determination module determining a phenotype of a tumor by using an ultrasound image; a second determination module determining at least one gene information related to breast cancer by using the phenotype of the tumor; a third determination module determining an individual breast cancer therapy by correlating the determined gene information with the gene information associated with the breast cancer therapy; and a display unit displaying a determination value determined by each of the determination modules.

10. The system of claim 9, wherein the ultrasound image includes a B-mode ultrasound image, a superb microvascular imaging (SMI) ultrasound image, and a contrast-enhanced ultrasound (CEUS) image.

11. The system of claim 10, wherein the phenotype determined through the B-mode ultrasound image includes size, shape, orientation, margin, and calcifications.

12. The system of claim 11, wherein the size is related to at least one gene selected from the group consisting of MIR941-1, IGLV6-57, HIST1H1B, HIST1H3I, ADH1B, PLIN4, and LUZP6; the shape is related to at least one gene selected from the group consisting of MIR941-1, ZFP36L1, SNHG9, H2AFY2, POTEI, UBE2Q2L, FABP7, IGHV3-43, IGKJ5, and IGKJ2; the orientation is related to at least one gene selected from the group consisting of TFF1, AREG, AGR3, TFF3, LINC00993, IGKV2-28, IGLV1-51, IGHV3-73, IGKV3-20, IGLV2-14, IGKV1-12, IGHV4-61, IGKV3D-15, IGHV1-3, IGHV4-4, IGHV1-18, IGHV4-34, IGHV3-74, CALML5, IGHJ5, and IGKJ2; the margin is related to HLA-C gene; and the calcifications are related to at least one gene selected from the group consisting of CALML3, HIST1H4F, IGHV4OR15-8, CCL19, and IGLV8-61.

13. The system of claim 10, wherein the phenotype determined through the SMI ultrasound image includes vascular index, vessel morphology, and penetrating vessel.

14. The system of claim 13, wherein the vascular index is related to at least one gene selected from the group consisting of IGHJ5, MIR1307, IGLV6-57, HLA-C, HIST2H2BE, CALML3, IGKV6-21, OR5P3, and MIR597; the vessel morphology is related to at least one gene selected from the group consisting of HIST1H4D, TUSC1, FZD8, NMI, IGF1R, UBB, SERHL2, NFIL3, CRIPAK, SNHG20, HBA2, and SNHG12; and the penetrating vessel is related to at least one gene selected from the group consisting of HIST1H4D, CST1, TRBC2, SLC25A2, KRT14, MFAP4, IGKV2-40, NFIL3, POTEE, POTEI, ALDH3B2, CRIPAK, IGHJ2, AREG, and IGKJ5.

15. The system of claim 10, wherein the phenotype determined through the contrast-enhanced ultrasound image includes enhancement order, enhancement margin, internal homogeneity, penetrating vessel, and perfusion defect.

16. The system of claim 15, wherein the enhancement order is related to at least one gene selected from the group consisting of IGKV1D-39, CCL3L3, IGHG4, IGKV1D-12, IGKV3D-11, SNHG12, CPB1, MIR562, and VTRNA2-1; the enhancement margin is related to at least one gene selected from the group consisting of STH, TFF1, STC2, AMY2A, HOXB5, IGKV1D-39, PHLDA2, HIST1H2AJ, TRAV14DV4, HIST1H1A, CXCL10, ISG15, IGHV4-39, IGKV3D-15, HIST2H2BF, HIST1H2BM, IGKV2-28, IGHV3-21, CALML5, IGHV1-18, IGKV2-29, IGHG4, IGHJ4, IGHJ5, and IGKJ2; the internal homogeneity is related to at least one gene selected from the group consisting of IGKJ5, HLA-DQA1, HIST1H1B, and IGHV3-74; the penetrating vessel is related to at least one gene selected from the group consisting of AGR2, HIST1H2BI, IGHV4-4, IGLV3-25, IGKV1D-39, IGHV1-2, IGHV3-15, IGKV1-27, IGLV3-1, IGKV2-40, IGKV2D-40, IGHV1-18, HIST1H2AG, IGHV3-33, IGKV1-12, IGKV1-17, IGHG1, TRBV5-6, IGHG4, IGHV4-61, IGKV2-28, IGHV1-8, IGHV4-39, IGHV3-21, IGHV3-9, IGKV3D-15, MIR562, IGHV1-69, IGHV4-31, IGHV1-3, and OR2J3; and the perfusion defect is related to at least one gene selected from the group consisting of HLA-DQA1, AREG, SNHG29, and IGHV3-74.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1A is a heat map image showing radiogenomic correlations according to the orientation at B-mode ultrasound imaging in 31 patients with breast cancer according to an example of the present disclosure. The heat map image shows 42 differentially expressed genes of log 2fc>2 or <−2. The rows represent individual tissue samples and the columns represent individual gene signature (Ensemble gene ID).

[0124] FIG. 1B is an ultrasound image showing cancer with parallel orientation in a 53-year-old patient according to an example of the present disclosure.

[0125] FIG. 1C is an ultrasound image showing cancer with non-parallel orientation in a 48-year-old patient according to an example of the present disclosure.

[0126] FIG. 1D is a volcano plot image showing radiogenomic correlations according to the orientation at B-mode ultrasound imaging in 31 patients with breast cancer according to an example of the present disclosure.

[0127] FIG. 2A is a heat map image showing radiogenomic correlations according to the presence of penetrating vessels at contrast agent-enhanced ultrasound in 31 patients with breast cancer according to an example of the present disclosure. The heat map image shows 52 differentially expressed genes of log 2fc>2 or <−2. The rows represent individual tissue samples and the columns represent individual gene signature (Ensemble green ID).

[0128] FIG. 2B is an ultrasound image showing cancer without penetrating vessel in a 50-year-old patient according to an example of the present disclosure.

[0129] FIG. 2C is an ultrasound image showing cancer with penetrating vessel in a 74-year-old patient according to an example of the present disclosure.

[0130] FIG. 2D is a volcano plot image showing radiogenomic correlations according to the presence of penetrating vessels at contrast agent-enhanced ultrasound imaging in 31 patients with breast cancer according to an example of the present disclosure.

[0131] FIGS. 3A and 3B are drawings showing interactions between the top 100 differentially expressed genes according to the orientation at B-mode ultrasound imaging according to an example of the present disclosure.

[0132] In FIG. 3A, the top network with a score of 29 including TFF1, TFF3, TP53, TGFBR2, AR, BAX, TERT, and POUF51 was associated with cell cycle, cellular growth and proliferation, and cancer. TFF1 and TFF3 were significantly upregulated genes showing direct interactions with TP53 and AR, respectively

[0133] In FIG. 3B, the second highest scoring network with a score of 21, including CCND1, AREG, NFKB1, CTNNB1, and RAC1, was associated with cancer, organismal survival, and injury.

DETAILED DESCRIPTION

[0134] Hereinafter, the present disclosure will be described in more detail by the following examples. However, these examples are used only for illustration, and the scope of the present disclosure is not limited by these examples.

Example 1: Patient Characteristics

[0135] This study was approved by the institutional review board. From January to October 2016, breast tumor patients (57 benign and 41 malignant) were included in a preliminary prospective study to assess association values between ultrasound parameters and histologic microvessel densities at vascular ultrasound imaging (SMI and CEUS) in the distinguishment of malignant from benign tumors. Of the 41 patients with malignant breast cancer, 31 patients (mean: 49.4 years old, range: 36-76 years old) who signed written informed consent to gene sequencing were included in the present study for correlation with vascular ultrasound imaging (SMI and CEUS) features of breast cancer. For 31 breast cancers (mean size: 21.8 mm, range: 7-48 mm), B-mode and vascular ultrasound (SMI and CEUS) images were observed. NGS was performed by using the whole genomic RNA obtained from surgically incised breast cancer tissues. Patient characteristics were summarized and shown in Table 1 below.

TABLE-US-00001 TABLE 1 Index — Number (percentage) B-mode ultrasound — — Size <20 mm 17 (54.8) ≥20 mm 14 (45.2) Shape Oval  5 (16.1) Irregular 26 (83.9) Echo pattern Isoechoic 3 (9.7) Heterogeneous 2 (6.5) Complex cystic and solid 1 (3.2) Hypoechoic 25 (80.6) Orientation Parallel 21 (67.7) Not parallel 10 (32.3) Margin Indistinct 13 (41.9) Angular  6 (19.4) Microlobulated  7 (22.6) Spiculated  5 (16.1) Calcifications Absent 18 (58.1) Present 13 (41.9) Superb microvascular imaging Vascular index <16.1% 19 (61.3) ≥16.1% 12 (38.7) Vessel morphology None or simple  6 (19.4) Complex 25 (80.6) Vessel distribution None or peripheral 2 (6.5) Central 29 (93.5) Penetrating vessel Absent  6 (19.4) Present 25 (80.6) Contrast-enhanced ultrasound Enhancement degree Hypo-enhancement 1 (3.2) Iso-enhancement 1 (3.2) Hyper-enhancement 29 (93.6) Enhancement order Diffuse  9 (29.0) Centripetal 22 (71.0) Enhancement margin Circumscribed 13 (41.9) Not circumscribed 18 (58.1) Internal homogeneity Homogeneous 17 (54.8) Heterogeneous 14 (45.2) Penetrating vessel Absent 11 (35.5) Present 20 (64.5) Perfusion defect Absent 20 (64.5) Present 11 (35.5) Pathology Tumor type Invasive ductal carcinoma 27 (87.1) Ductal carcinoma in situ  4 (12.9) Immunohistochemical result ER positive 21/31 (67.7)   PR positive 22/31 (71.0)   HER2 positive 8/31 (25.8)   ER = estrogen receptor, PR = progesterone receptor, HER2 = human epidermal growth factor receptor 2

[0136] In 16 ultrasound imaging phenotypes, the analyses for echo pattern at B-mode ultrasound, vessel distribution at SMI, and enhancement order at contrast-enhanced ultrasound were excluded since the numbers of samples of two groups were not balanced and data were skewed to one group between the two to result in low statistical reliability.

Example 2: Ultrasound Imaging and Analyses

[0137] Aplio 500 system (Canon Medical Systems, Tokyo, Japan) with a 5-14 MHz linear transducer was used. The ultrasound examinations were performed by a radiologist with 18 years of experience in breast examinations. Two radiologists (with 12 and 5 years of experience in breast imaging, respectively) analyzed the imaging phenotypes according to breast imaging reports and data systems: B-mode phenotypes included size (20 mm vs<20 mm), shape (irregular vs oval or round), echo pattern (complex cystic and solid or hypoechoic vs isoechoic or heterogeneous), orientation (not parallel vs parallel), margin (angular, microlobulated, or spiculated vs indistinct), and calcifications (present vs absent).

[0138] After B-mode ultrasound evaluation, SMI and CEUS were performed. At SMI, the plane with the richest vessels was stored as a representative image for evaluation. CEUS was performed immediately after SMI. The contrast agent (SonoVue (Bracco, Milan, Italy)) was mixed with saline and injected in a bolus fashion, and video clips were recorded during continuous scanning. The imaging parameters for SMI were velocity scale<3 cm/s, dynamic range 21 dB, and frame rate 27-60 frames/s. Those for CEUS were mechanical index 0.08, frame rate 10 frames/s, gain 80, and dynamic range 65 dB.

[0139] The SMI phenotypes included vascular index (%, the ratio between the pixels for the Doppler signal and those for the whole lesion, the mean vascular index of <31 cancers vs mean vascular index), vessel morphology (complex (branching or shunting) vs none or simple (dot-like or linear)), distribution (central (vessel detected within the lesion) vs none or peripheral (all vessels located at the margin)), and penetrating vessels (present vs absent).

[0140] The CEUS phenotypes included enhancement degree (hyperenhancement vs iso- or hypoenhancement), order (centripetal vs centrifugal or diffuse), margin (uncircumscribed vs circumscribed), internal homogeneity (heterogeneous vs homogeneous), penetrating vessels (present vs absent), and perfusion detection (present vs absent).

Example 3: RNA Sequencing and Analyses

[0141] Total RNA concentration was calculated by using Quant-IT RiboGreen (Invitrogen, USA) and 100 ng was subjected to sequencing library construction. The mRNA-seq library was prepared using the paired-end mRNA sequencing sample preparation kit (TruSeq RNA access library kit, Illumina, USA). By using the Illumina HiSeq 2500 sequencing system, paired-end sequencing (2×100 bp) was performed. Low-quality and adapter sequences from produced paired-end reads were trimmed using the Trim Galore software (version 0.5.0) and Cutadapt (version 1.18).

[0142] The genome analysis toolkit, which was the best practice for workflow for SNP and InDel calling on RNA sequencing data, was used. Briefly, the spliced transcripts alignment to a reference 2-pass method was used to align trimmed reads to the human reference genome (hg19). Picard command line tools were used to process SAM files produced in the above-mentioned step to add read group information, sorting, marking duplicates, and indexing. Variants were called and filtered using the genome analysis toolkit tools HaplotypeCaller and VariantFiltration. RNA variants were annotated using ANNOVAR. Functional enrichment analysis and pathway analysis were performed by using ingenuity pathway analysis software (Ingenuity Systems, USA).

Example 4: Correlations Between Ultrasound Imaging Phenotypes and Gene Expressions

[0143] The genes expressed differentially between two groups of each ultrasound imaging phenotype were detected by using Tablemaker and Ballgown. First, Tablemaker (version 2.1.1) was used to estimate fragments per kilobase of transcript per million mapped reads (FPKM) for each assembled transcript. Then, the results from Tablemaker were integrated into the software environment R (version 3.5.0) by using the R package Ballgown (version 2.10.0). Ballgown was used to calculate differential gene expression from RNA sequencing data. FPKM was used to estimate the level of gene expression, and the P value for differential expression was extracted by using a parametric F-test comparing nested linear models. The log 2 fold change (log 2fc) of the gene expression between two groups was calculated by using the Ballgown “stattest” function. Differential gene expression results were visualized by using a volcano plot and heat map in R. The results are shown in FIGS. 1 and 2.

Conclusion

Genes Expressed Differentially According to Ultrasound Imaging Phenotypes

[0144] A total of 340 genes were expressed differentially according to the ultrasound imaging phenotypes with the standard of P<0.05 and log 2fc>2 or <−2: 92 genes were upregulated and 263 were downregulated. Of these, 228 were noncoding RNA with unknown function or pseudogenes, and the other 112 were protein-coding genes (n=102) or noncoding RNA with known function (microRNA (n=5), snoRNA (n=4), and lncRNA (n=1)).

[0145] Tables 2 to 14 show the summary of 112 significantly up- or down-regulated genes according to ultrasound imaging phenotypes. Twenty-seven of the 112 genes have been reported as being relevant to breast cancer in terms of tumor growth, invasion, metastasis, and drug resistance. (*: genes reported as being relevant to breast cancer)

TABLE-US-00002 TABLE 2 Gene Symbol Gene Name Log2fc P Value MIR941-1 MicroRNA 941-1 4.04 <0.01 IGLV6-57 Immunoglobulin Lambda Variable 6-57 2.44 0.02 HIST1H1B Histone Cluster 1 H1 Family Member B 2.05 0.03 HIST1H3I Histone Cluster 1 H3 Family Member I 2.01 0.01 ADH1B Alcohol Dehydrogenase 1B (Class I), Beta Polypeptide −2.24 <0.01 PLIN4 Perilipin 4 −2.40 0.01 LUZP6 Leucine Zipper Protein 6 −3.15 0.02
Genes expressed differentially according to ultrasound imaging phenotype—B-mode ultrasound_Size

TABLE-US-00003 TABLE 3 Gene Symbol Gene Name Log2fc P Value MIR941-1 MicroRNA 941-1 4.64 0.01 ZFP36L1* ZFP36 Ring Finger Protein Like 1 2.08 <0.01 SNHG9 Small Nucleolar RNA Host Gene 9 2.00 0.02 H2AFY2 H2A Histone Family Member Y2 −2.03 <0.01 POTEI POTE Ankyrin Domain Family Member I −2.10 0.01 UBE2Q2L Ubiquitin Conjugating Enzyme E2 Q2 Like −2.13 0.04 FABP7* Fatty Acid Binding Protein 7 −2.36 0.01 IGHV3-43 Immunoglobulin Heavy Variable 3-43 −2.76 0.05 IGKJ5 Immunoglobulin Kappa Joining 5 −7.82 0.01 IGKJ2 Immunoglobulin Kappa Joining 2 −9.27 0.03
Genes expressed differentially according to ultrasound imaging phenotype—B-mode ultrasound_Shape

TABLE-US-00004 TABLE 4 Gene P Symbol Gene Name Log2fc Value TFF1* Trefoil Factor 1 4.00 <0.01 AREG* Amphiregulin 2.58 <0.01 AGR3* Anterior Gradient 3, Protein Disulphide 2.57 <0.01 Isomerase Family Member TFF3* Trefoil Factor 3 2.47 <0.01 LINC00993* Long Intergenic Non-Protein Coding 2.05 0.02 RNA 993 IGKV2-28 Immunoglobulin Kappa Variable 2-28 −2.01 0.04 IGLV1-51 Immunoglobulin Lambda Variable 1-51 −2.02 0.04 IGHV3-73 Immunoglobulin Heavy Variable 3-73 −2.10 <0.01 IGKV3-20 Immunoglobulin Kappa Variable 3-20 −2.18 0.02 IGLV2-14 Immunoglobulin Lambda Variable 2-14 −2.20 0.03 IGKV1-12 Immunoglobulin Kappa Variable 1-12 −2.25 0.03 IGHV4-61 Immunoglobulin Heavy Variable 4-61 −2.28 0.03 IGKV3D-15 Immunoglobulin Kappa Variable 3D-15 −2.31 0.03 IGHV1-3 Immunoglobulin Heavy Variable 1-3 −2.40 0.05 IGHV4-4 Immunoglobulin Heavy Variable 4-4 −2.44 0.03 IGHV1-18 Immunoglobulin Heavy Variable 1-18 −2.46 0.02 IGHV4-34 Immunoglobulin Heavy Variable 4-34 −2.60 0.01 IGHV3-74 Immunoglobulin Heavy Variable 3-74 −2.70 0.01 CALML5 Calmodulin Like 5 −2.98 0.02 IGHJ5 Immunoglobulin Heavy Joining 5 −5.60 0.03 IGKJ2 Immunoglobulin Kappa Joining 2 −7.12 0.05
Genes expressed differentially according to ultrasound imaging phenotype—B-mode ultrasound_Orientation

TABLE-US-00005 TABLE 5 Gene Symbol Gene Name Log2fc P Value HLA-C Major Histocompatibility 2.13 0.01 Complex, Class I, C
Genes expressed differentially according to ultrasound imaging phenotype—B-mode ultrasound_Margin

TABLE-US-00006 TABLE 6 Gene Symbol Gene Name Log2fc P Value CALML3 Calmodulin Like 3 −2.03 0.02 HIST1H4F Histone Cluster 1 H4 Family −2.03 0.01 Member F IGHV4OR15-8 Immunoglobulin Heavy −2.05 0.02 Variable 4/OR15-8 CCL19 C-C motif chemokine ligand 19 −2.19 0.01 IGLV8-61 Immunoglobulin Lambda −2.53 0.03 Variable 8-61
Genes expressed differentially according to ultrasound imaging phenotype—B-mode ultrasound_Calcifications

TABLE-US-00007 TABLE 7 Gene Symbol Gene Name Log2fc P Value IGHJ5 Immunoglobulin Heavy Joining 5 5.80 0.02 MIR1307* MicroRNA 1307 3.53 <0.01 IGLV6-57 Immunoglobulin Lambda Variable 2.49 0.01 6-57 HLA-C Major Histocompatibility Complex, 2.21 0.01 Class I, C HIST2H2BE* Histone Cluster 2 H2B Family 2.05 <0.01 Member E CALML3 Calmodulin Like 3 −2.25 <0.01 IGKV6-21 Immunoglobulin Kappa Variable 6-21 −2.37 0.02 OR5P3 Olfactory Receptor Family 5 −2.37 <0.01 Subfamily P Member 3 MIR597* MicroRNA 597 −2.64 0.05
Genes expressed differentially according to ultrasound imaging phenotype—SMI_Vascular index

TABLE-US-00008 TABLE 8 Gene Symbol Gene Name Log2fc P Value HIST1H4D Histone Cluster 1 H4 Family Member D 2.68 0.01 TUSC1 Tumor Suppressor Candidate 1 2.55 <0.01 FZD8* Frizzled Class Receptor 8 2.02 0.01 NMI* N-Myc And STAT Interactor −2.02 <0.01 IGF1R* Insulin Like Growth Factor 1 Receptor −2.03 0.01 UBB* Ubiquitin B −2.05 0.01 SERHL2 Serine Hydrolase Like 2 −2.06 0.03 NFIL3 Nuclear Factor, Interleukin 3 Regulated −2.12 <0.01 CRIPAK* Cysteine Rich PAK1 Inhibitor −2.42 0.01 SNHG20* Small Nucleolar RNA Host Gene 20 −2.42 0.04 HBA2 Hemoglobin Subunit Alpha 2 −2.57 0.05 SNHG12* Small Nucleolar RNA Host Gene 12 −3.11 0.01
Genes expressed differentially according to ultrasound imaging phenotype—SMI_Vessel morphology

TABLE-US-00009 TABLE 9 Gene Symbol Gene Name Log2fc P Value HIST1H4D Histone Cluster 1 H4 Family Member D 3.45 <0.01 CST1* Cystatin SN 2.65 <0.01 TRBC2 T Cell Receptor Beta Constant 2 2.22 0.01 SLC25A2 Solute Carrier Family 25 Member 2 −2.00 0.01 KRT14 Keratin 14 −2.02 0.02 MFAP4 Microfibril Associated Protein 4 −2.06 <0.01 IGKV2-40 Immunoglobulin Kappa Variable 2-40 −2.07 0.05 NFIL3 Nuclear Factor, Interleukin 3 Regulated −2.12 0.01 POTEE POTE Ankyrin Domain Family −2.35 0.02 Member E POTEI POTE Ankyrin Domain Family −2.39 <0.01 Member I ALDH3B2 Aldehyde Dehydrogenase 3 Family −2.40 0.01 Member B2 CRIPAK* Cysteine Rich PAK1 Inhibitor −2.57 0.01 IGHJ2 Immunoglobulin Heavy Joining 2 −3.01 0.04 AREG* Amphiregulin −3.03 0.01 IGKJ5 Immunoglobulin Kappa Variable 1D-39 −6.58 0.02
Genes expressed differentially according to ultrasound imaging phenotype—SMI_Penetrating vessel

TABLE-US-00010 TABLE 10 Gene Symbol Gene Name Log2fc P Value IGKV1D-39 Immunoglobulin Kappa Variable 1D-39 −2.01 0.03 CCL3L3 C-C Motif Chemokine Ligand 3 Like 3 −2.06 0.03 IGHG4 Immunoglobulin Heavy Constant −2.08 0.03 Gamma 4 (G4m Marker) IGKV1D-12 Immunoglobulin Kappa Variable 1D-12 −2.15 0.03 IGKV3D-11 Immunoglobulin Kappa Variable 3D-11 −2.25 0.02 SNHG12* Small Nucleolar RNA Host Gene 12 −2.54 <0.01 CPB1 Carboxypeptidase B1 −2.71 <0.01 MIR562* MicroRNA 562 −3.19 <0.01 VTRNA2-1* Vault RNA 2-1 −5.85 0.01
Genes expressed differentially according to ultrasound imaging phenotype—Contrast-enhanced ultrasound_Enhancement order

TABLE-US-00011 TABLE 11 Gene Symbol Gene Name Log2fc P Value STH Saitohin 3.12 <0.01 TFF1* Trefoil Factor 1 2.95 0.02 STC2* Stanniocalcin 2 2.66 <0.01 AMY2A Amylase, Alpha 2A (Pancreatic) 2.28 <0.01 HOXB5* Homeobox B5 2.17 <0.01 IGKV1D-39 Immunoglobulin Kappa Variable 1D-39 −2.01 0.02 PHLDA2* Pleckstrin Homology Like Domain −2.01 <0.01 Family A Member 2 HIST1H2AJ Histone Cluster 1 H2A Family −2.04 0.03 Member J TRAV14DV4 T Cell Receptor Alpha Variable −2.06 <0.01 14/Delta Variable 4 HIST1H1A Histone Cluster 1 H1 Family Member A −2.08 0.01 CXCL10* C-X-C Motif Chemokine Ligand 10 −2.08 0.01 ISG15 ISG15 Ubiquitin-Like Modifier −2.16 0.01 IGHV4-39 Immunoglobulin Heavy Variable 4-39 −2.19 0.02 IGKV3D-15 Immunoglobulin Kappa Variable 3D-15 −2.25 0.03 HIST2H2BF Histone Cluster 2 H2B Family −2.25 0.01 Member F HIST1H2BM Histone Cluster 1 H2B Family −2.25 0.03 Member M IGKV2-28 Immunoglobulin Kappa Variable 2-28 −2.29 0.02 IGHV3-21 Immunoglobulin Heavy Variable 3-21 −2.52 0.02 CALML5 Calmodulin Like 5 −2.66 0.03 IGHV1-18 Immunoglobulin Heavy Variable 1-18 −2.79 0.01 IGKV2-29 Immunoglobulin Kappa Variable 2-29 −2.83 <0.01 IGHG4 Immunoglobulin Heavy Constant −2.89 0.01 Gamma 4 (G4m Marker) IGHJ4 Immunoglobulin Heavy Joining 4 −6.92 0.02 IGHJ5 Immunoglobulin Heavy Joining 5 −7.60 <0.01 IGKJ2 Immunoglobulin Kappa Joining 2 −8.61 0.01
Genes expressed differentially according to ultrasound imaging phenotype—Contrast-enhanced ultrasound_Enhancement margin

TABLE-US-00012 TABLE 12 Gene Symbol Gene Name Log2fc P Value IGKJ5 Immunoglobulin Kappa Joining 5 4.65 0.05 HLA-DQA1* Major Histocompatibility Complex, 3.29 <0.01 Class II, DQ Alpha 1 HIST1H1B Histone Cluster 1 H1 Family Member B 2.02 0.05 IGHV3-74 Immunoglobulin Heavy Variable 3-74 −2.37 0.01
Genes expressed differentially according to ultrasound imaging phenotype—Contrast-enhanced ultrasound_Internal homogeneity

TABLE-US-00013 TABLE 13 Gene Symbol Gene Name Log2fc P Value AGR2* Anterior Gradient 2, Protein Disulphide 2.09 0.01 Isomerase Family Member HIST1H2BI Histone Cluster 1 H2B Family −2.05 0.02 Member I IGHV4-4 Immunoglobulin Heavy Variable 4-4 −2.05 0.05 IGLV3-25 Immunoglobulin Lambda Variable 3-25 −2.06 0.04 IGKV1D-39 Immunoglobulin Kappa Variable 1D-39 −2.09 0.02 IGHV1-2 Immunoglobulin Heavy Variable 1-2 −2.12 0.03 IGHV3-15 Immunoglobulin Heavy Variable 3-15 −2.13 0.03 IGKV1-27 Immunoglobulin Kappa Variable 1-27 −2.13 0.03 IGLV3-1 Immunoglobulin Lambda Variable 3-1 −2.15 0.03 IGKV2-40 Immunoglobulin Kappa Variable 2-40 −2.16 0.04 IGKV2D-40 Immunoglobulin Kappa Variable 2D-40 −2.23 0.01 IGHV1-18 Immunoglobulin Heavy Variable 1-18 −2.23 0.01 HIST1H2AG Histone Cluster 1 H2A Family −2.26 0.01 Member G IGHV3-33 Immunoglobulin Heavy Variable 3-33 −2.29 0.02 IGKV1-12 Immunoglobulin Kappa Variable 1-12 −2.34 0.01 IGKV1-17 Immunoglobulin Kappa Variable 1-17 −2.34 0.01 IGHG1 Immunoglobulin Heavy Constant −2.36 0.02 Gamma 1 (G1m Marker) TRBV5-6 T Cell Receptor Beta Variable 5-6 −2.39 <0.01 IGHG4 Immunoglobulin Heavy Constant −2.40 0.02 Gamma 4 (G4m Marker) IGHV4-61 Immunoglobulin Heavy Variable 4-61 −2.41 0.03 IGKV2-28 Immunoglobulin Kappa Variable 2-28 −2.41 0.02 IGHV1-8 Immunoglobulin Heavy Variable 1-8 −2.47 0.03 IGHV4-39 Immunoglobulin Heavy Variable 4-39 −2.48 0.01 IGHV3-21 Immunoglobulin Heavy Variable 3-21 −2.61 0.01 IGHV3-9 Immunoglobulin Heavy Variable 3-9 −2.91 0.04 IGKV3D-15 Immunoglobulin Kappa Variable 3D-15 −3.09 <0.01 MIR562* MicroRNA 562 −3.11 0.01 IGHV1-69 Immunoglobulin Heavy Variable 1-69 −3.21 0.01 IGHV4-31 Immunoglobulin Heavy Variable 4-31 −3.21 <0.01 IGHV1-3 Immunoglobulin Heavy Variable 1-3 −3.28 0.01 OR2J3 Olfactory Receptor Family 2 −3.52 0.05 Subfamily J Member 3
Genes expressed differentially according to ultrasound imaging phenotype—Contrast-enhanced ultrasound_Penetrating vessel

TABLE-US-00014 TABLE 14 Gene Symbol Gene Name Log2fc P Value HLA-DQA1* Major Histocompatibility Complex, 2.80 <0.01 Class II, DQ Alpha 1 AREG* Amphiregulin −2.02 0.02 SNHG29 Small Nucleolar RNA Host Gene 29 −2.09 0.01 IGHV3-74 Immunoglobulin Heavy Variable 3-74 −2.17 0.02
Genes expressed differentially according to ultrasound imaging phenotype—Contrast-enhanced ultrasound Perfusion defect

[0146] Of the B-mode ultrasound imaging phenotypes, orientation showed the most upregulated genes related to breast cancer. Breast cancers with nonparallel orientation showed overexpression of TFF1, AREG, AGR3, TFF3, and LINC00993 compared with those with parallel orientation. FIG. 1 shows gene expression data according to orientation by using a heat map and a volcano plot.

[0147] At SMI, the complex vessel morphology of cancer was related to the upregulation of FZD8 and the downregulation of IGF1R, NMI, and CRIPAK. The presence of a penetrating vessel at SMI was related to the upregulation of CST1 and the downregulation of CRIPAK. Elevated vascular index was related to the upregulation of MIR1307 and HIST2H2BE and the downregulation of MIR597.

[0148] At CEUS, the enhancement order was related to the downregulation of SNHG12 and VTRNA2-1. The enhancement margin was related to the upregulation of TFF1, STC2, and HOXB5 and the downregulation of PHLDA2. At the CEUS examination, the presence of the penetrating vessel was related to the upregulation of AGR2. FIG. 2 shows gene expression data according to the presence of penetrating vessel at CEUS by using a heat map and a volcano plot.

Gene Network Identification

[0149] The top 100 differentially expressed genes according to each ultrasound imaging phenotype were used to identify gene networks based on the Ingenuity Pathways Knowledge Base. FIG. 3 shows the top two networks according to the orientation on B-mode ultrasound imaging. The top network with a score of 29 including TFF1, TFF3, TP53, TGFBR2, AR, BAX, TERT, and POUF51 genes was associated with cell cycle, cellular growth and proliferation, and cancer. TFF1 and TFF3 show direct interactions with TP53 and AR, respectively. The second highest scoring network with a score of 21 including AREG, CCND1, NFKB1, and RAC1 genes was associated with cancer, organismal survival, and injury.

[0150] The top network of penetrating vessels at CEUS images with a score of 18 included genes of AGR2, EGFR, EGR1, FOXA1, Mek, MAPK1, and AREG and was associated with cellular movement, organismal survival, and cell cycle.

[0151] The top network of vessel morphology at SMI with a score of 13 included genes of IGF1R, ESR1, IRS-1, RARA, FOXO1, NR3C1, HDAC2, and histone h3 and the functions thereof were associated with cell death and survival, and cancer.

Gene Function Analysis

[0152] Enriched functional annotations of expression profiles were obtained from ingenuity pathway analysis with an input of the top 100 differentially expressed genes according to each ultrasound imaging phenotype. The functions associated with general cancer or breast cancer were mainly related to the orientation on B-mode ultrasound imaging and the vessel morphology at SMI, and the results are shown in Table 3.

[0153] Enriched functions of significantly upregulated genes (TFF3, TFF1, and AREG) in nonparallel cancers included transformation of carcinoma cells, bending of DNA, cell movement, migration of cells, activation of epithelial cells, and mitosis of epithelial cell lines. Enriched functions of significantly up- or down-regulated genes (FZD8, IGF1R, and CRIPAK) in cancer with complex vessel morphology included proliferation of epithelial cells, repair of DNA, attachment, aggregation, anoikis, and growth inhibition.

TABLE-US-00015 TABLE 15 Functional Annotation P Value Molecules Transformation of <0.0032 TFF3 carcinoma cells Bending of DNA 0.0097 TFF1 Cell movement 0.0105 AREG, ELMO1, IGHV4-34, IGKV1-12, IGKV2-28, IGKV3-20, IGLV1-51, IGLV2-14, PARD6B, SLC12A6, TFF1, TFF3, TNFRSF1A, TNFRSF1B Migration of cells 0.0235 AREG, ELMO1, IGHV4-34, IGKV1-12, IGKV2-28, IGKV3-20, IGLV1-51, IGLV2-14, PARD6B, TFF1, TNFRSF1A, TNFRSF1B Activation of 0.0288 AREG epithelial cells Mitosis of epithelial 0.0414 AREG cell lines
Functional analysis of differentially expressed genes according to ultrasound phenotype (orientation)

TABLE-US-00016 TABLE 16 Functional Annotation P Value Molecules Apoptosis of adenocarcinoma <0.0102 IGF1R, SNHG20 cell lines Cell proliferation of <0.0112 IGF1R, SNHG20 adenocarcinoma cell lines Depression of RNA <0.0114 UBB Attachment of breast cancer <0.0142 IGF1R cell lines Aggregation of breast cancer <0.0170 IGF1R cell lines Polyploidization of tumor 0.0111 IGF1R cell lines Transformation of carcinoma 0.0111 IGF1R cell lines Repair of DNA 0.0161 HIST1H4D, IGF1R, UBB Double-stranded DNA 0.0185. HIST1H4D, IGF1R break repair 0.0252 FZD8, IGF1R Proliferation of epithelial cells Anoikis of breast cancer 0.0262 IGF1R cell lines Signal transduction 0.0265 CCR3, GNAT3, HTR1A, IGF1R, RXFP2 Breast cancer 0.0311 ATAD2B, CRIPAK, FZD8, GLI2, HBA1/ HBA2, HIST1H4D, IGF1R, NFIL3, PCSK5 Global genomic repair 0.0357 UBB Contact growth inhibition of 0.0371 IGF1R breast cancer cell lines
Functional annotation of differentially expressed genes according to ultrasound phenotype (vessel morphology)

Analysis of Pathways Associated with Genes and Diseases of Interest

[0154] To determine significantly changed signal pathways, canonical pathway analyses were performed on the basis of functional annotation of the top 100 differentially expressed genes according to each ultrasound imaging phenotype by using corresponding bibliographic data. Ingenuity pathways technical data were used as a reference set. As shown in Table 4, several canonical pathways associated with cancer or breast cancer were identified regarding orientation and vessel morphology.

[0155] The AREG associated with orientation at B-mode ultrasound imaging was related to human HER2 signaling in breast cancer and ErbB signaling in canonical pathway analysis. The FZD8 or NMI associated with vessel morphology at SMI was related to Wnt/b-catenin signaling, PCP pathway, Wnt/Ca+pathway, prolactin signaling, regulation of epithelial-mesenchymal transition pathway, and molecular mechanisms of cancer.

TABLE-US-00017 TABLE 17 Ingenuity Canonical Pathway −log (P value) Ratio Molecules HER-2 Signaling in 1.41 0.0208 PARD6B, Breast Cancer AREG Breast Cancer Regulation 0.824 <0.0195 CALML5, by Stathmin1 PPM1J ErbB Signaling 0.538 <0.0195 AREG
Canonical pathway of differentially expressed genes according to ultrasound phenotype (orientation)

TABLE-US-00018 TABLE 18 −log (P Ingenuity Canonical Pathway value) Ratio Molecules PTEN Signaling 1.9 0.0163 IGF1R, MAGI3 Wnt/β-catenin Signaling 1.63 0.0118 FZD8, UBB DNA Methylation and 1.33 0.0294 HIST1H4D Transcriptional Repression Signaling PCP pathway 1.09 0.0167 FZD8 Wnt/Ca+ pathway 1.09 0.0164 FZD8 Toll-like Receptor Signaling 1 0.0133 UBB Myc Mediated Apoptosis Signaling 0.991 0.013 IGF1R Growth Hormone Signaling 0.943 0.0116 IGF1R Estrogen-Dependent Breast 0.943 0.0116 IGF1R Cancer Signaling Prolactin Signaling 0.921 0.011 NMI GABA Receptor Signaling 0.91 0.0106 UBB NER Pathway 0.876 <0.0198 HIST1H4D IGF-1 Signaling 0.833 <0.0190 IGF1R RhoA Signaling 0.807 <0.0183 IGF1R STAT3 Pathway 0.772 <0.0176 IGF1R Hereditary Breast Cancer Signaling 0.724 <0.0167 UBB NF-κB Signaling 0.642 <0.0154 IGF1R Regulation of the Epithelial- 0.62 <0.0151 FZD8 Mesenchymal Transition Pathway NRF2-mediated Oxidative 0.613 <0.0150 UBB Stress Response EIF2 Signaling 0.575 <0.0146 IGF1R Molecular Mechanisms of Cancer 0.375 <0.0126 FZD8
Canonical pathway of differentially expressed genes according to ultrasound phenotype (vessel morphology)

Determination of Therapy Through Correlation Between Ultrasound Phenotypes and Genes

[0156] Patients with the upregulation of TFF1, TFF3, AREG, and AGR3 genes among the breast cancer patients with non-parallel orientation at B-mode ultrasound were subjected to hormone therapy as targeted therapy for estrogen receptor (ER). The patients who received treatment showed good therapy response without recurrence at 4 years of follow-up after therapy.

CONCLUSION

[0157] At B-mode ultrasound, it was identified that the nonparallel orientation of the cancer showed the upregulation of TFF1, TFF3, AREG, and AGR3. These genes were enriched in ER—positive breast cancers in the previous study. Additionally, TFF1, TFF3, and AGR3 were significantly associated with low tumor grades and proposed as early serum markers of breast cancer. From the results, nine (90%) of 10 cancers with nonparallel orientation revealed ER positivity, whereas 11 (52.4%) of 21 parallel cancers exhibited ER positivity. These results suggest that the nonparallel orientation at ultrasound might be associated with ER positivity and low tumor grades. AREG is involved in regulating ErbB signaling and may be a new target for breast cancer treatment. Our study similarly found that ErbB and HER-2 signals were related to AREG in canonical pathway analyses.

[0158] However, the action of TFF1 and TFF3 on disease prognosis is controversial. TFF1 and TFF3 are known to enhance stromal and lymphovascular invasion, resulting in increasing toxicity to lymph node metastases and distant metastases. From the results, the top network of the orientation including genes of TFF1 and TFF3 was associated with cell growth and proliferation. Therefore, these results are likely related to cancer progression and metastasis.

[0159] The complex vessel morphology including branches or branching vessels of tumor tissue is one of the malignant vascular features and might reflect tumor angiogenesis. From the results, complex vessel morphology at SMI was associated with the downregulation of the gene of CRIPAK and the upregulation of the gene of FZD8. Both the genes are related to tumor angiogenesis. CRIPAK is a negative regulator of PAK1, which promotes angiogenesis through increased permeability and vascular cell migration. In addition, PAK1 was related to tamoxifen therapy and resistance to tumor recurrence, and therefore is receiving attention as a therapeutic target. FZD8 was associated with the top network including VEGFA, a most significant gene in tumor angiogenesis. Another study reported that FZD8 plays a key role in drug resistance in triple-negative breast cancer through FZD8-mediated Wnt signaling and may be a potential target for improving therapeutic efficacy. Complex vessel morphology was associated with the downregulation of IGF1R, which is associated with low tumor grades, low metastasis capacity, and high overall survival. This study supported that the top networks of vessel morphology were associated with cancer and cell death. Therefore, complex vessel morphology in ultrasound images may be a predictor of tumor angiogenesis, tamoxifen therapy for luminal breast cancer, or chemotherapy and drug prognosis for triple-negative breast cancers.

[0160] Other vascular ultrasound imaging phenotypes were also associated with breast cancer-related genes. Penetrating vessels at SMI and CEUS were related to the upregulation of CST1 and AGR2, which promote breast cancer cell proliferation, metastasis, and low survival rates. Elevated vascular index at SMI was related to the upregulation of MIR1307 and HIST2H2BE, which are associated with drug resistance of breast cancer, and the downregulation of MIR597, which inhibits breast cancer cell proliferation and invasion. At CEUS, the centripetal enhancement of breast cancer was related to the downregulation of SNHG12 and VTRNA2-1, which act as tumor inhibitors. At CEUS, the uncircumscribed breast cancer margin was related to the upregulation of TFF1 and HOXB5, which are associated with metastasis or invasion of breast cancer, and the downregulation of PHLDA2, a tumor inhibition gene.