Rapid and Accurate Diagnosis of Breast Cancer by Fine-Needle Aspiration Biopsy Using the “Click-to-Sense” Method

doi:10.21203/rs.3.rs-5249777/v1

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Rapid and Accurate Diagnosis of Breast Cancer by Fine-Needle Aspiration Biopsy Using the “Click-to-Sense” Method

https://doi.org/10.21203/rs.3.rs-5249777/v1

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We have previously demonstrated the value of the “click-to-sense” (CTS) assay, in which a fluorescent probe targeting acrolein can detect cancer cells and differentiate between malignant and benign lesions in breast tissue. In this study, we assessed the usefulness of the CTS assay for diagnosis of breast tumors by fine-needle aspiration biopsy (FNAB). A total of 126 FNABs were performed on live tissue samples obtained by surgery (63 breast cancers, 31 benign breast tumors, and 32 normal breast glands). CTS reagents (CTS probe and Hoechst dye mixed with encapsulating agents) were added to the aspirated cells and placed on slides, which were then cover-slipped and imaged under a fluorescence microscope. Another FNAB slide was prepared for each of the same live tissue samples, fixed in ethanol, and subjected to Papanicolaou (PAP) staining. The diagnostic accuracy of the CTS assay was compared with that of PAP staining by histopathological examination of permanent sections. The CTS assay had a sensitivity of 92.1%, a specificity of 96.8%, and an accuracy of 94.4% (119/126 samples); the respective values for PAP staining were 98.4%, 89.8%, and 94.2% (114/121 samples). The insufficiency/inadequacy rate was 0% for the CTS assay and 4% for PAP staining (5/126 samples). In conclusion, the CTS assay is as accurate as PAP staining for FNAB of breast lesions. This assay could potentially replace PAP staining because it has a lower inadequacy rate and is simpler and less labor-intensive and time-consuming to perform.

mammary neoplasms

biopsy

fine-needle

click-to-sense

acrolein

Breast cancer is the leading malignancy in women in terms of both incidence and mortality [1], and early detection and accurate diagnosis of breast lesions are important in terms of subsequent therapeutic strategies [2]. The two most common techniques used for pathological diagnosis of breast lesions are core needle biopsy (CNB) for histological examination and fine-needle aspiration biopsy (FNAB) for cytopathological examination, both of which have specific advantages and limitations [3]. Histological diagnosis by CNB provides detailed information over and above cell morphology, including tissue structure and intercellular relationships, thereby offering higher diagnostic accuracy in comparison with FNAB [3]. However, CNB is an invasive procedure that must be performed under local anesthesia and imposes a significant burden on patients in terms of discomfort, a long processing time, and high cost [4].

In contrast, FNAB with Papanicolaou (PAP) staining is a simpler, less expensive procedure with a lower risk of complications and a high patient acceptance rate owing to its non-invasiveness and causes minimal discomfort [4,5].However, unlike histological diagnosis by CNB, FNAB only evaluates the morphology and characteristics of cells, thereby providing limited pathological insights and occasionally yielding false-positive or false-negative results [4].In a meta-analysis of 12 studies (including 1802 samples) that compared the diagnostic accuracy of FNAB with that of CNB for breast tumors, FNAB demonstrated a sensitivity of 74% (95% confidence interval 72–77) and a specificity of 96% (95% confidence interval 94–98) [3,6-8]. Another report mentioned a high insufficiency/inadequacy rate of 17.7% with FNAB, which was attributed in part to the masses being too small for adequate collection of cells [9].Other potential reasons for insufficient/inadequate samples include esiccation of cells and poor fixation [10].Although the recently devised International Academy of Cytology Yokohama system is now used for breast FNAB with consistent reporting methods and clear definitions for each category [11,12].FNAB does not guarantee absolute accuracy or definitively exclude cancer cells from breast tissue samples, even when they are absent. For initial evaluation of breast tumors, a comprehensive approach known as “triple assessment” is recommended, which involves clinical examination, breast imaging (mammography and/or ultrasound), and pathological evaluation [5]. Obtaining accurate results with FNAB also requires specialists to have appropriate technical skills and experience [13,14].

We have developed a “click-to-sense” (CTS) probe that detects acrolein, a substance released from cancer cells under oxidative stress that can be visualized by fluorescence microscopy [15,16]. Using live tissue samples resected from patients with breast cancer, Tanei et al. and Kubo et al. demonstrated that this CTS probe can rapidly, selectively, and sensitively differentiate between a cancerous breast lesion and a normal glandular or benign proliferative breast lesion and requires staining of live tissue for only 5 min [17,18].The CTS probe is an “in-cell” reactivity probe for detection of acrolein by fluorescence based on an acrolein/azide click reaction and can visualize in detail the morphology of the various types of cancer cells in which acrolein accumulates [19].Therefore, the CTS assay has potential for rapid cytopathological assessment of live cells obtained via FNAB. Although there have been reports of the ability of a fluorescent probe to distinguish between living cancer cells and normal cultured cells in vitro [20-22], there have been no reports on use of this novel diagnostic approach in live cells from patients.

In this study, we developed a CTS assay using live cells obtained for breast tumors via FNAB and compared its diagnostic accuracy with that of PAP staining of live tissue obtained from the same lesion by histopathological examination of permanent sections. The aim of this research was to determine whether the CTS assay can accurately distinguish between malignant and benign/normal breast tissues obtained by FNAB.

Patients and breast tumors

The study included 126 breast tissue samples from 89 patients who were retrospectively identified to have undergone benign tumorectomy (63 samples), or lumpectomy or mastectomy for primary breast cancer without neoadjuvant systemic therapy (63 samples) at Osaka University Hospital between March 2022 and January 2024 (Table 1). Two benign tumors were resected in one patient, and three benign tumors were resected at the same time as mastectomy for breast cancer. Thirty-three normal breast gland tissue samples were obtained from distant breast tissue in the patients with breast cancer who underwent mastectomy. In total, FNAB examinations were performed for 126 breast tissue samples (63 breast cancers, 30 benign breast tumors, and 33 normal breast glands) and subjected to both the CTS assay and PAP staining. The breast tissues were fixed in 10% buffered formalin, embedded in paraﬀin, and diagnosed by histopathological examination of permanent sections (PS analysis).

Reagents

Four micrograms of the CTS probe were dissolved in 1 µL of dimethyl sulfoxide. Next, 4 µL of Hoechst 33342 dye (Dojindo, Kumamoto, Japan), 4 µL of Hoechst 33258 dye (Dojindo), and 490 µL of phosphate-buffered saline were added to create a CTS probe solution with a final concentration of 12 µM. For each analysis, 25 µL of the CTS probe was combined with 25 µL of encapsulating agent and placed on a glass slide. ProLong Live Antifade Reagent (Thermo Fisher Scientific, Fresno, CA, USA).

Synthesis of the CTS probe

The CTS probe was synthesized using a method that has been reported elsewhere [16]. First, 5(6)-TAMRA-succinimidyl ester (101.8 mg, 0.193 mmol, 1.0 eq) was added to 2,6-diisopropyl-4-aminomethyl aniline (39.8 mg, 0.193 mmol, 1.0 eq) in dimethylformamide (2.0 mL). The reaction mixture was then stirred under a nitrogen atmosphere for 24 hours. The resulting solution was concentrated to dryness under reduced pressure to give the precursor CTS as a black-purple crude compound (Supplementary Fig. 1). ESI-HRMS m/z calcd for C₃₈H₄₃N₄O₄ ([M+H]⁺) 619.3279, found 619.3275.

The crude compound was then dissolved in acetic acid and water in a ratio of 9 to 1 (2.0 mL). Next, sodium nitrite (41.4 mg, 0.600 mmol, 3.11 eq) and sodium azide (36.7 mg, 0.565 mmol, 2.93 eq) were slowly added to the solution at 0°C. After stirring for 30 min, the solution was concentrated to dryness under reduced pressure. The mixture was purified by reversed-phase high-pressure liquid chromatography (RP-HPLC) to give the second-generation CTS probe (in TFA salt form) as a dark purple solid (92.8 mg, 64% in two steps). The RP-HPLC conditions were as follows: column, Cosmosil 5C₁₈-AR300 (Nacalai Tesque, Inc., Kyoto, Japan), 20 × 250 mm; mobile phase A, 0.1% TFA in H₂O; mobile phase B, 0.1% TFA in CH₃CN; gradient elution, 0–4 min at 85% B, 4–9 min at 85%–100% B, and 9–10 min at 100% B; flow rate, 10 mL/min; and UV detection at 254 nm. ESI-HRMS m/z calcd for C₃₈H₄₁N₆O₄([M+H]⁺) 645.3184, found 645.3186. ¹H NMR (400 MHz, CD₃CN) δ 8.76–7.39 (m, 3H), 7.21 (d, J = 33.1 Hz, 2H), 7.11 (dd, J = 9.5, 4.9 Hz, 2H), 6.92 (d, J = 9.1 Hz, 2H), 6.84 (s, 2H), 4.54 (dd, J = 33.0, 5.4 Hz, 2H), 3.36 (tq, J = 13.7, 6.8 Hz, 2H), 3.24 (s, 12H), 1.23 (dd, J = 22.2, 6.8 Hz, 12H).

Mechanism via which the CTS probe detects acrolein

We also determined the mechanism via which acrolein can be visualized in cancer cells using the CTS probe (Supplementary Fig. 1). Our findings indicate that the CTS probe is taken up by cells via endocytosis, with the TAMRA scaffold playing an important role in internalization. In healthy cells, the CTS probe remains inactive and is excreted back into the extracellular environment by exocytosis. However, in cancer cells, the CTS probe undergoes a [3+2] cycloaddition reaction upon encountering intracellular acrolein, resulting in formation of a diazo derivative. This derivative then reacts with the nearest organelle in the cancer cells, leading to covalent attachment of the TAMRA fluorophore to the organelle [19], allowing for labeling and imaging of cancerous tissue at the cellular level.

CTS assay

We used the CTS assay to analyze 126 breast tissue samples as well as fluorescence microscopy analysis using both CTS probe fluorescence staining (red) and Hoechst 33342 and 33258 fluorescence staining (blue) (Fig. 1). Each live breast tissue sample was obtained by FNAB using a 22-G needle and a 10-mL syringe. The contents of the syringe were then placed on a glass slide, after which 50 μL of the CTS probe with Hoechst dye and encapsulating agent were immediately dripped onto the slide. Finally, a glass coverslip was applied and the slide was imaged using a fluorescence microscope. Whole-slide fluorescence images with a range of 2 × 1 cm were captured in a low-power field (40×). Images of cell clusters stained red by the CTS probe were also captured in high-power fields (200×, range, 5 mm × 5 mm/400×, range, 2.5 mm × 2.5 mm) for morphological examination (Supplementary Fig. 2). The mean time required for diagnosis using the CTS assay was 5 min per tissue sample, and only one person was required to perform the assay.

Analysis of CTS images

CTS images were analyzed in accordance with the diagnostic criteria outlined in the CTS assay flow chart (Fig. 2) and categorized into four groups. Representative fluorescence and PAP stained images for each category are shown in Fig. 2. Category 4 was diagnosed as malignant (positive) and categories 1–3 as benign or normal (negative). In Figure 2, representative fluorescence microscopy images obtained by the CTS assay and by PAP staining revealed invasive ductal carcinoma (category 4), fibroadenoma (category 2–3), a mucocele-like lesion (category 2), and normal breast tissue (category 1). PAP staining identified the invasive ductal carcinoma as malignant (Fig. 2B4) and the fibroadenoma and normal breast tissue as benign (Fig. 2B1–2B3).

In category 4, fluorescent aggregated cells (≥5) were red in the cytoplasm with enlarged nuclei (≥10 μm) and had a high nuclear/cytoplasmic (N/C) ratio and irregular nucleus. We investigated the major axis of the nuclei of cells stained with Hoechst dye (blue) (Fig. 2A4). The criterion for defining cells with enlarged nuclei (≥10 μm) as malignant was based on an examination of the major nuclear axis of 20 cancer cells and 20 normal or benign ductal epithelial cells stained with Hoechst dye (blue) in which the CTS assay established 10 μm as the threshold for differentiation between malignant and benign/normal cells (Supplementary Fig. 3).

In category 3, although fluorescent aggregated cells (≥5) were red in the cytoplasm, they had a low nuclear cytoplasmic (N/C) ratio and smaller nuclei, were <10 μm in size, and had a regular shape (Fig. 2A3). In category 2, epithelial cells without fluorescent aggregated cells (<4) were detected (Fig. 2A2). In category 1, the CTS assay did not identify any epithelial cells with cytoplasmic staining (Fig. 2A1).

Three blinded diagnosticians working independently visually evaluated each CTS image to determine whether it was positive or negative. CTS images were deemed to be positive when diagnosed by two or three diagnosticians as positive using the CTS assay and to be negative when diagnosed as positive by zero or one diagnostician. The diagnosticians registered their findings in the Research Electronic Data Capture (Redcap) system hosted at Osaka University using a HIPAA-compliant secure web application [23].Access to servers and systems is restricted by user accounts and passwords, and a full audit trail is recorded.

Papanicolaou staining

We performed FNAB twice for each lesion. The second samples were fixed in ethanol and subjected to PAP staining, after which they were compared with the samples subjected to the CTS assay (Fig. 1). The PAP staining protocol was performed following the same procedure as that used in the Pathology Department at Osaka University. PAP staining was interpreted by skilled experienced cytopathologists and specialists using International Academy of Cytology Yokohama system-compliant categories.

Measurement of fluorescence intensity of the CTS assay

Regions of cellular aggregation were excised from the CTS images and analyzed by Nikon NIS-Elements image analysis software with 200× magnification. The median fluorescence intensity of the areas that were stained red was measured in each cell.

Statistical analysis

The diagnostic accuracy of the CTS assay was compared with that of PAP staining by PS analysis. The McNemar test was used to assess the relationship between the results of the CTS assay and those for PAP staining. The Mann–Whitney test was used to compare the fluorescence intensity of the CTS assay between malignant and benign/normal breast tissue. All statistical analyses were performed using R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). A P-value <0.05 was deemed to be statistically significant.

Ethics statement

The study protocol was approved by the medical ethics committee of Osaka University (approval number 20197). In view of the retrospective observational nature of the research, the requirement for written informed consent was waived. However, informed consent was obtained via the opt-out route on the hospital’s website.

Microscopic fluorescence images of the CTS assay

When using the CTS assay, cells were identified from whole-slide fluorescence images, and images of cell clusters stained red by the CTS probe were observed under high magnification for examination of cell morphology (Supplementary Fig. 2). Representative CTS assay-positive and PAP staining-positive images from invasive ductal cancer, ductal carcinoma in situ, invasive lobular carcinoma, or mucinous carcinoma are shown in Figure 3. The cytoplasm of malignant cells was stained with TAMRA fluorescence dye derived from the CTS probe (red), and the nuclei of these cells were stained with Hoechst dye (blue). The malignant cells clearly showed morphological features similar to those observed with PAP staining and PS analysis.

Representative CTS assay-negative and PAP staining-negative images from benign lesions and normal breast tissue are shown in Figure 4. CTS images of epithelial cells from benign lesions, such as intraductal papilloma, phyllodes tumor, ductal hyperplasia, and fibroadenoma, detected minimal or weak red cytoplasm that was less intense than that of malignant cells (Fig. 4A1–4D1). CTS images of normal breast tissues were detected with only nuclei of no red cytoplasmic cells (Fig. 4E1). There were clear differences in morphological features and boundaries between malignant and benign/normal cells.

Comparison of the CTS assay and PAP staining with PS analysis

The results of the CTS assay and PAP staining analysis are compared with those of the PS analysis in Table 2. The results of the CTS assay and PAP staining are shown according to histological type in Supplementary Table 1. As shown in Figure 5, 60 samples were classified as positive (category 4) by the CTS assay, 58 of the 60 samples were diagnosed as malignant by PS analysis, and two were classified as benign/normal (accuracy, 96.7%). Twenty-seven samples were classified as category 3 (benign/normal, n=24; malignant, n=3; accuracy, 88.9%). Thirteen samples were classified as category 2 (benign/normal, n=11; malignant, n=2; accuracy, 84.6%). All 26 samples classified as category 1 were benign/normal on PS analysis (accuracy, 100%). PAP staining had an insufficiency/inadequacy rate of 4.0% (5/126 samples). In contrast, none of the 126 samples were insufficient/inadequate for the CTS assay. Finally, the diagnostic accuracy of the CTS assay was 94.4% with a sensitivity of 92.1% and a specificity of 96.8%. Meanwhile, the diagnostic accuracy of the PAP staining was 94.2% with a sensitivity of 98.4% and a specificity of 89.8% after exclusion of insufficient/inadequate samples (Table 2). There was no significant difference in diagnostic accuracy between the CTS assay and PAP staining analysis (p=0.8, McNemar's test).

False-positive and false-negative CTS assay samples

There were two false-positive CTS assay samples, one of which was intraductal papilloma and the other was fibroadenoma with epithelial cells that stained faintly red in the CTS assay. However, there were few red-stained cells without nuclear enlargement (Supplementary Fig. 4). The CTS assay also yielded five false-negative results (invasive ductal cancer, n=2; invasive lobular carcinoma, n=1; ductal carcinoma in situ, n=1; mucinous carcinoma, n=1; Supplementary Fig. 5). Cells in the two invasive ductal cancer samples and in the invasive lobular carcinoma sample strongly stained red in the CTS assay. However, these cells had small, non-irregular nuclei, leading to misclassification as negative.

Red fluorescence intensity was measured in regions of cell aggregation on CTS images of 126 samples with 200× magnification. We found that red staining of CTS images was much stronger in breast cancer cells than in cells from benign/normal breast tissue. There were significant differences in red fluorescence intensity on CTS images for malignant cells among each group (Mann–Whitney test). There was a statistically significant difference in fluorescence intensity between malignant and benign/normal breast tissue (P<0.0001, Fig. 6).

We have previously found the CTS assay to be effective for rapid differential diagnosis of malignant and benign/normal breast lesions, suggesting a potential application for this assay in FNAB of breast tumors. In this study, we compared the accuracy of the CTS assay with that of standard PAP staining by observing cells obtained from malignant and benign tumors and from normal breast tissues by FNAB. We performed FNAB twice on the live tissue samples and evaluated each slide using the CTS assay and PAP staining. We also standardized the morphological examination visualized by the CTS assay and developed a flow chart showing the diagnostic criteria that should be used when performing this assay (Fig. 2). Finally, we were able to obtain a very high accuracy rate of 94.4% (119/126 samples) between the CTS assay and PS analysis when performed in a blinded manner, which was comparable with the rate of 94.2% (114/121 samples) between the PAP staining and PS analyses. In particular, sensitivity was lower and specificity was higher for the CTS assay than for the PAP staining analysis. The number of individuals diagnosed as positive by three independent diagnosticians is shown in Supplementary Table 2. A significant proportion (57%) of CTS-positive samples were consistently diagnosed as positive by all three diagnosticians, while the majority (80%) of CTS-negative samples were consistently diagnosed as negative by the same three diagnosticians.

We encountered two false-positives and five false-negatives when using the CTS assay. The two false-positives were intraductal papilloma and fibroadenoma, which showed lightly red-stained epithelial cells in the CTS assay (Supplementary Fig. 4). Two of the three diagnosticians deemed these benign lesions to be positive, even though there were usually few red-stained cells without nuclear enlargement, indicating the presence of benign cells with high oxidative stress stained in red by the CTS assay. The five false-negatives included two cases of invasive ductal cancer, one of invasive lobular carcinoma, one of ductal carcinoma in situ, and one of mucinous carcinoma. Two of the three diagnosticians diagnosed these four malignancies as negative. The sample with ductal carcinoma in situ showed few cancer cells on PAP staining and was diagnosed as a single-duct ductal carcinoma in situ by PS analysis, and the CTS assay result was a consequence of poor cell sampling in FNAB (Supplementary Fig. 5A1–5A3). Although there were cells that strongly stained red in the CTS assay for two samples of invasive ductal cancer and one of invasive lobular carcinoma, these cells were small and had non-irregular nuclei, which led to misclassification as negative (Supplementary Fig. 5B1–5B3, 5C1–5C3, 5D1–5D3). The mucinous carcinoma sample showed weak staining and had small nuclei (Supplementary Fig. 5E1–5E3), leading to a benign diagnosis because the CTS reagent became diluted after excessive application of liquid, which reduced the intensity of red fluorescence.

Furthermore, none of the samples were considered insufficient/inadequate for the CTS assay, but 4% were found to be insufficient/inadequate for PAP staining analysis. This difference may be explained by the fact that unlike PAP staining, the CTS assay does not include fixing in ethanol, which is associated with desiccation of cells and poor fixation.

Use of the CTS assay for FNAB requires only simple techniques, namely, application of the CTS probe, Hoechst dye, and encapsulating agent to a glass slide containing aspirated cells followed by cover-slipping. In contrast, the PAP staining process is complex and must be performed by an expert cytopathologist. The CTS assay has the advantages of high specificity, no risk of insufficient/inadequate sampling, and being simple to perform, time-saving, and less labor-intensive in comparison with PAP staining analysis. In clinical practice, it takes at least a few days to report the PAP staining results. In contrast, the results of the CTS assay are available after 5 min. Rapid onsite evaluation systems can now triage FNAB samples using ultrasound guidance, although a cytopathologist is still needed for FNAB analysis [24-26]. Breast FNAB and PAP staining analysis require specific training in technique and slide interpretation for accurate reporting and involves a steep learning curve [14].Meanwhile, the CTS assay can visualize cancer cells without the need for a pathologist. The workload for this assay entails checking the fluorescence images for red-stained cells with irregular nuclear contours or nuclear enlargement. However, unlike PAP staining, the CTS assay does not provide details on internal nuclear structure.

The limitations of this study include its single-center design and the limited sample size. The potential utility of our method for FNAB using the CTS assay requires assessment in larger multicenter clinical trials.

This study demonstrates that the CTS assay makes a significant contribution to cytopathological diagnosis of live cancer cells and normal cells obtained from patients, despite previous reports indicating that this assay is only useful for diagnosis of cultured living cells in vitro [20-22].

Research is underway at our center to develop an artificial intelligence (AI)-assisted diagnostic imaging system for use with the CTS assay, which may represent an advance on the cytopathological diagnostic methods available. We are currently conducting a collaborative research project with the Graduate School of Information Science and Technology at Osaka University in the hope of developing a program that uses AI-driven techniques for diagnosis of breast cancer on fluorescence images obtained using the CTS assay. Recent advances in AI are transforming the diagnosis of breast cancer on hematoxylin–eosin-stained slides, significantly improving the accuracy, efficiency, and consistency of PS analysis. Several innovative AI-based tools are commercially available, offering pathologists and oncologists valuable support when diagnosing breast cancer [27]. The first use of AI in a pathology laboratory was in cytopathology when a computer-assisted PAP staining screening method was created [28]. AI technologies have the potential to completely transform cytopathology. In a cervical screening study, Kurita et al. demonstrated that AI could classify images of liquid-based cervical cytology samples as malignant or normal and that AI would allow cytologists or cytopathologists to obtain support from their counterparts worldwide [29]. We speculate that the CTS assay could potentially be applied to cytology for other cancers, such as cervical carcinoma, and we are presently conducting a clinical trial of the CTS assay for cervical cytology of PAP smears.

In summary, the CTS assay offers diagnostic accuracy comparable with that of PAP staining analysis in FNAB of breast tumors. It is simpler and less labor-intensive and time-consuming than PAP staining analysis. This assay may eventually replace PAP staining analysis for outpatient cytopathological diagnosis of breast lesions. Currently, the CTS assay is evaluated visually. However, in the future, diagnosis may be possible using AI-based images, which would be a significant advance in cytopathological diagnostic methodology. We also believe that there is potential for application of cytopathological diagnosis to other cancerous lesions in the future.

We have shown that the CTS assay is as accurate as PAP staining in FNAB for breast tumors, suggesting it could potentially replace PAP staining analysis. The insufficiency/inadequacy rate is lower for the CTS assay than for PAP staining. Furthermore, the CTS assay is simpler, less labor-intensive, and less time-consuming to perform.

Conflicts of Interest and Source of Funding

JSPS KAKENHI (Grant Numbers JP22K08709, JP24K11762, and JP24K01625), AMED (Grant Number JP22ck0106783), AMED FORCE and the Canon Foundation.

Author Contribution

Y.K. and T.T. wrote the main manuscript text and figures, and R.A. prepared only supplementary figures 1. All authors reviewed the manuscript."

Acknowledgement

This research was conducted using the Research Electronic Data Capture system operated by the Osaka University Graduate School of Medicine and the Osaka University Hospital. The authors thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

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Table 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

Supplementaryfigure1.tif
Supplementary Fig. 1. Synthesis of the CTS probe and the mechanism via which it visualizes acrolein. Abbreviation: CTS, click-to-sense
Supplementaryfigure2.tif
Supplementary Fig. 2. Fluorescence images captured in the CTS assay. Whole-slide fluorescence images (area, 2 × 1 cm) captured at low magnification (40×). Images of cell clusters stained red by the CTS probe were also captured at high magnification (200×, area 5 mm × 5 mm/400×, area 2.5 mm × 2.5 mm) for morphological examination. Abbreviation: CTS, click-to-sense
Supplementaryfigure3.tif
Supplementary Fig. 3. Major axis of nuclei. The major axes of 20 cancer cells and 20 normal ductal epithelial cells were measured in the CTS images. A threshold of 10 µm was established in the CTS assay to differentiate between malignant and benign/normal cells. Abbreviation: CTS, click-to-sense
Supplementaryfigure4.tif
Supplementary Fig. 4. Representative images of CTS assay false-positive samples. CTS images were classified as positive by the CTS assay (A1–B1). On PAP stain analysis, A2 was classified as negative and B2 as positive. In contrast, the histopathological images (A3–E3) were diagnosed as intraductal papilloma (A1–3) and fibroadenoma (B1–3). Abbreviations: CTS, click-to-sense; PAP, Papanicolaou; PS, permanent section
Supplementaryfigure5.tif
Supplementary Fig. 5. Representative images of CTS assay false-negative samples. CTS images were classified as negative by the CTS assay (A1–E1). On PAP stain analysis, A2–E2 was classified as positive. In contrast, histopathological images (A3–E3) were diagnosed as low-grade DCIS (A1–3), IDC (B1–3, C1–3), ILC (D1–3), and mucinous carcinoma (E1–3). Abbreviations: CTS, click-to-sense; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; PAP, Papanicolaou; PS, permanent section
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Rapid and Accurate Diagnosis of Breast Cancer by Fine-Needle Aspiration Biopsy Using the “Click-to-Sense” Method

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