Clinical Significance of Hepatitis A Virus Cellular Receptor 1 (HAVCR-1) in Breast Cancer

Clinical Significance of Hepatitis A Virus Cellular Receptor 1 (HAVCR-1) in Breast Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Significance of Hepatitis A Virus Cellular Receptor 1 (HAVCR-1) in Breast Cancer Xiaoshan Cao, Binbin Cong, Wen G. Jiang, Hong Liu, Tracey A Martin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7939575/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Hepatitis A virus cellular receptor 1 (HAVCR-1), first discovered as the entry factor for hepatitis A virus, has since been recognized as a tumor-associated antigen and is currently being intensively explored as a prognostic biomarker across multiple malignancies, including gastric and colorectal cancers. However, its contribution to breast cancer remains ambiguous. This study investigated the expression and clinical significance of HAVCR-1 in breast cancer. Methods: HAVCR-1 expression was evaluated via quantitative real-time PCR (qPCR) and immunohistochemistry (IHC) in a cohort of fresh-frozen normal and breast cancer tissues. Expression levels were correlated with clinicopathological parameters, including the Nottingham prognostic index, tumor stage, histological subtype, hormone receptor status (ER, PR, HER2), and survival outcomes. The prognostic and predictive value of HAVCR-1 was assessed via chi-square tests and receiver operating characteristic (ROC) analysis. The median follow-up period was 120 months. In vitro, HAVCR-1 was knocked down via siRNA in the MCF-7 and MDA-MB-231 cell lines, and the response to docetaxel was evaluated on the basis of the IC50 values. Additionally, ROC plotter analysis was used to assess the association between HAVCR-1 expression and therapeutic response in breast cancer patients. Results: Although qPCR revealed no statistically significant difference in HAVCR-1 mRNA levels between tumor and normal tissues (P=0.2697), IHC revealed increased protein expression in breast cancer tissues. Low HAVCR-1 expression was significantly associated with improved overall survival (OS, P < 0.01) and relapse-free survival (RFS, P=0.04). Subgroup analysis indicated that the survival benefit of low HAVCR-1 expression was particularly evident in ER-positive (P=0.046) and HER2-positive (P=0.004) subtypes but not in triple-negative breast cancer (TNBC). In vitro knockdown of HAVCR-1 conferred resistance to docetaxel in both MCF-7 (IC50: 0.86 nM vs. 1.75 nM) and MDA-MB-231 (IC50: 1.35 nM vs. 15.48 nM) cells. Clinically, patients with high HAVCR-1 expression are more likely to exhibit resistance to chemotherapy, particularly those with the luminal A, luminal B, and TNBC subtypes, but not those with HER2+/ER− tumors. Conclusion: HAVCR-1 is an independent prognostic factor for OS and RFS in breast cancer patients and may serve as a predictive biomarker for chemotherapy response. Its combination with ER, PR, and HER2 status could enhance prognostic stratification and therapeutic decision-making. HAVCR-1 breast cancer overall survival relapse-free survival chemotherapy response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Currently, the incidence of breast carcinoma is increasing worldwide, and its impact continues to widen [ 1 – 3 ] . More than 2.3 million fresh diagnoses and approximately 685 000 deaths were recorded in 2020, and demographic aging alone is expected to increase these numbers beyond 3 million cases and 1 million deaths annually by 2040 [ 2 ] . Breast cancer continues to have a large impact on the number of cancer deaths worldwide [ 2 ] . Once tumor cells disseminate to distant organs, the disease becomes largely incurable [ 4 – 5 ] ; hence, there is an urgent need for fresh molecular flags and druggable targets that can anticipate or blunt progression. The hepatitis A virus entry factor HAVCR-1—also annotated as T-cell immunoglobulin mucin-1 (TIM-1) or human kidney injury molecule-1 (KIM-1)—was first identified as the portal for hepatotropic picornaviruses, yet its transcriptional territory extends far beyond the liver [ 6 ] . Robust baseline expression has been documented in the kidney, testis, small bowel, colon and spleen, implying broader physiological roles [ 6 ] . Accumulating evidence links HAVCR-1 to aggressive solid tumors: clear-cell renal carcinoma [ 7 ] , cervical cancer [ 8 ] , gastric adenocarcinoma [ 9 ] and colorectal cancer [ 10 ] all display gain-of-expression signatures that correlate with adverse stages and outcomes, positioning the receptor as a candidate oncogenic driver. Furthermore, functional assays have shown that HAVCR-1 promotes cell motility, matrix invasion and anchorage-independent growth [ 8 , 10 ] . while its presence on activated T and NK cells modulates cytokine release and immune surveillance—properties that could sculpt the tumor microenvironment [ 11 – 13 ] . However, to the best of our knowledge, the expression of HAVCR-1 and its precise biological function in breast cancer are ambiguous. On the basis of these observations and previous studies, we hypothesized that HAVCR-1 could play a role in the occurrence and development of breast cancer. To verify this hypothesis, we evaluated the expression of HAVCR-1 in breast cancer and the relationship between HAVCR-1 expression and the prognosis of patients with HAVCR-1. 2. Materials and methods 2.1 Breast cancer tissue collection A total of 127 breast cancer specimens and 33 adjacent histologically normal tissues were harvested intraoperatively, snap-frozen in liquid nitrogen, and stored until analysis; all donors provided written informed consent. The study protocol was approved by the Bro Taf Local Research Ethics Committee. Clinicopathological data were extracted from reports verified by a specialist pathologist, and postoperative surveillance was maintained for a median duration of 120 months. 2.2 RNA extraction, reverse transcription and quantitative polymerase chain reaction (PCR) The breast tissue pieces and cell line pellets were homogenized in AB Gene Total RNA reagent (Advanced Biotechnologies, UK). The RNA yield and 260/280 purity ratios were recorded on a WPA UV-1101 spectrophotometer (Biotech Photometer, UK). First-strand cDNA was synthesized from 0.25 µg of RNA with a Sigma reverse-transcription kit (Poole, UK) and checked with podoplanin (PDPLN) primers. HAVCR-1 transcript numbers were quantified with the Amplifluor UniPrimer system (InterGen, UK) on an iCycler IQ thermal cycler (Bio-Rad, USA). The reactions contained Abgene qPCR MasterMix and were run at 95°C for 15 min, followed by 65 cycles at 95°C for 15 s, 55°C for 30 s, and 72°C for 20 s. A plasmid dilution series generated copy number standards; β-actin served as the endogenous control, and cytokeratin-19 (CK19) was used to correct for the epithelial content. The primer/probe sequences are provided in Table 1 ; detailed protocols were reported previously [ 14 ] . Table 1 Primers and probes for real-time quantitative PCR molecule Forward Primers (5’→3’) Reverse Primers (5’→3’) GAPDH CTGAGTACGTCGTGGAGTC ACTGAACCTGACCGTACA CAGAGATGATGACCCTTTTG HAVCR-1 GACAATGACTGTTTCAACGA ACTGAACCTGACCGTACA TGGAGGAACAAAGGTAGAGA Cytokeratin 19 CAGGTCCGAGGTTACTGAC ACTGAACCTGACCGTACA CACTTTCTGCCAGTGTGTCTTC Note: The Z sequence is highlighted in bold. 2.3 Immunohistochemical staining of breast samples Six-micron cryosections were prepared from snap-frozen tumors and matched background tissue (Leica DMB cryostat, Milton Keynes, UK), collected on SuperFrost Plus slides, and fixed in ice-cold acetone for 15 min. After brief air-drying and rehydration, nonspecific binding was blocked with 0.6% bovine serum albumin for 20 min. The sections were incubated for 1 h with HAVCR-1 primary antibody (70R-14161) or with antibody diluent alone (negative control), followed by three Tris-buffered saline washes. Biotinylated secondary antibody was applied for 30 min, the signal was amplified with VECTASTAIN ABC reagent (Vector Laboratories, USA), and the color was developed with diaminobenzidine (5 min, dark). The slides were counterstained with Gill’s hematoxylin, dehydrated through graded methanol, cleared in xylene, and coverslipped. Immunoreactivity was evaluated independently by two investigators. 2.4 Cell culture MCF-7 and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Sigma, Poole, UK) supplemented with 10% fetal bovine serum and 1% penicillin‒streptomycin at 37°C in a humidified 5% CO₂ atmosphere. 2.5 HAVCR-1 knockdown (KD) HAVRC-1 was knocked down in the MCF-7 and MDA-MB-231 cell lines through siRNA (sc-61691 Santa Cruz Biotechnology Inc., USA) transfection. For optimal transfection efficiency, Santa Cruz Biotechnology’s siRNA transfection reagent (sc-29528), siRNA transfection medium (sc-36868) and siRNA dilution buffer (sc-29527) are recommended. 2.6 Docetaxel cytotoxicity assays To test whether HAVCR-1 modulates docetaxel tolerance, wild-type and HAVCR-1-silenced MCF-7 and MDA-MB-231 cells were plated at 5 × 10³ per well (96-well format) in 100 µL of medium containing a 2-log docetaxel gradient (MCF-7: 0.02–2 000 nM; MDA-MB-231: 0.48–48 000 nM) or drug-free control (six replicates). After 72 h at 37°C/5% CO₂, the medium was removed, the cells were gently rinsed with PBS, fixed with 4% paraformaldehyde (10 min, room temperature), and stained with 0.5% crystal violet (10 min). After these plates were dried, the dye was solubilized in 10% (v/v) acetic acid (100 µL/well), and the absorbance was read at 595 nm on an LT-4500 microplate reader. Survival curves were constructed in Microsoft Excel. 3. Statistical analysis Transcript levels of HAVCR-1 between two groups were compared with two-tailed Student’s t test under normality or the Mann–Whitney U test when normality was rejected. For > 2 groups, one-way ANOVA plus Tukey’s post hoc test was applied to normally distributed data; otherwise, the Kruskal–Wallis test followed by Dunn’s correction was used. Association strength and predictive value were examined via χ² tests and receiver operating characteristic (ROC) curve analysis in SPSS 27 (IBM UK, Porto). Plots were generated with GraphPad Prism 9.5 (San Diego, CA). A two-sided P < 0.05 was considered significant and is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. 4. Results 4.1 Expression of HAVCR-1 in breast cancer HAVCR-1 mRNA was quantified by qPCR in a Cardiff cohort of breast carcinomas paired with adjacent normal tissue. Although median levels were elevated in tumors, the change did not reach statistical significance (Table 2 ), and no associations emerged with standard clinicopathological variables. When ductal carcinomas were stratified by grade, Grade 1 cases presented markedly higher HAVCR-1 transcript counts than Grade 2 or Grade 3 lesions did (P < 0.05, Fig. 1 ). Among patients who ultimately died from breast cancer, those with ER-positive tumors presented significantly greater HAVCR-1 expression than those with ER-negative primaries did (P < 0.05, Fig. 2 ); in contrast, no difference was observed between those who remained disease-free and those who developed progression (Table 2 ). Table 2 Transcript expression profile of HAVCR-1 in comparison with the clinicopathological information of the Cardiff breast cancer cohort. Kruskal‒Wallis ANOVA on RANKS. Characteristic Sample number (n) Median transcript expression Q1 Q3 P-value Normal 33 1 0 145 0.2697 a Tumor 127 21 0 316 Nottingham prognostic index 2.4- 5.4 16 1 0 131 Tumor Grade 1 24 123 2 8316 0.098 b 2 43 13 0 173 3 58 13 0 228 TNM stage 1 70 18 0 413 0.894 b 2 40 13 0 365 3 7 45.1 0 47.8 4 4 107 0 15004 Clinical outcome Disease free 90 42 0 379 0.253 b With Metastasis 7 0 0 429 With local recurrence 5 183 0 544 Died of breast cancer 16 10 0 1093 Nottingham prognostic index in ductal breast cancer 2.4- 5.4 12 18 0 221 TNM in ductal breast cancer 1 54 40 0 702 0.817 b 2 32 17 0 330 3 6 46.4 11.3 47.8 4 2 107 * * Clinical outcome in ductal breast cancer Disease free 69 46 0 466 0.291 b With Metastasis 7 0 0 429 With local recurrence 4 0 0 365 Died of breast cancer 11 21 0 1348 Note: a Mann‒Whitney test; b Kruskal‒Wallis ANOVA on RANKS 4.2 Immunohistochemical staining of HAVCR-1 expression in clinical breast cancer tissue Historical fresh frozen breast tissue sections from a set of samples previously available in our laboratory were utilized to explore HAVCR-1 protein expression levels. Representative micrographs (Fig. 3 ) show consistently denser tumor-associated immunostaining relative to adjacent normal parenchyma. 4.3 Impact of HAVCR-1 expression on relapse-free survival and overall survival in breast cancer patients To evaluate the prognostic value of HAVCR-1, we constructed relapse-free survival (RFS) and overall survival (OS) curves for the Cardiff series using the cohort-specific median transcript level as the cutoff. Patients below the median (low HAVCR-1, n = 85) had significantly better RFS (mean = 125.0 months) than those with high HAVCR-1 (n = 13) (mean = 87.8 months) (P < 0.001). Analysis of the effect of HAVCR-1 expression on the HER-2-positive subtype (Fig. 4 C) revealed that patients who expressed low levels of HAVCR-1 (n = 41) had significantly better RFS than those who expressed high levels of HAVCR-1 (P = 0.003). The trends in the ER-positive subtype and TNBC subtype were similar to those in the HER2 subtype, without statistical significance (P > 0.05). The relationship between HAVCR-1 expression and patient OS was also explored in the Cardiff cohort (Fig. 5 ). Patients who presented with low HAVCR-1 levels (n = 85) had significantly better OS (mean = 141.5 months) than did those with high HAVCR-1 levels (n = 13) (mean = 91.5 months) (P < 0.001, Fig. 5 A). In the subgroup analysis, high HAVCR-1 expression was associated with worse OS in the ER-positive (P = 0.038, Fig. 5 B) and HER-2-positive subtypes (P = 0.002, Fig. 5 C). 4.4 Expression and knockdown of HAVCR-1 HAVCR-1 levels were significantly lower in HAVCR-1-KD MDA-MB-231 (Fig. 6 A) and MCF-7 (Fig. 6 B) cells than in WT cells at the mRNA level, confirming that HAVCR-1-KD cell models were successfully established with breast cancer cells. 4.5 HAVCR-1 KD enhances breast cancer cell resistance to docetaxel Following the incubation of the cellular models in serially diluted docetaxel for 72 hours, the loss of HAVCR-1 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (the IC50 values for WT and HAVCR1 KD were 0.86 nM and 1.75 nM for MCF-7 cells and 1.35 nM and 15.48 nM for MDA-MB-231 cells, respectively) (Fig. 7 ). 4.6 HAVCR-1 as a predictor of chemotherapy response in the breast cancer ROC plotter cohort To examine whether HAVCR-1 modulates resistance to systemic therapy, we mined the ROC plotter resource ( http://www.rocplot.org ), a compendium of 3 104 breast cancer transcriptomes with annotated drug-response calls [ 15 ] . The response was defined according to the Response Evaluation Criteria in Solid Tumors 1.1 criteria. Transcript levels between responders and nonresponders were visualized as box-and-whisker plots and compared with the Mann–Whitney U test; ROC curves were generated to quantify the predictive accuracy of HAVCR-1 for both chemotherapy and targeted agents (Fekete & Győrffy, 2019). 4.6.1 All-chemotherapy response on the basis of pathological complete response Probe set 207052_at was used to compare HAVCR-1 transcript levels with pathological response after any neoadjuvant chemotherapy across all ROC-plotter subtypes (responders, n = 532; nonresponders, n = 1100) (Fig. 8 ). As shown in Fig. 8 A, patients who responded to chemotherapies had significantly lower median HAVCR-1 expression than nonresponders did (median 36 vs 90, P = 8.7x10 −−18 ). The beneficial effects of low-level HAVCR1 and patient sensitivity to chemotherapies were observed in the luminal A subtype (5 vs 74, P = 3.7x10 − 19 ; Fig. 8 B), luminal B subtype (median 112 vs 139, P = 0.00062; Fig. 8 C) and TNBC subtype (median 32 vs 63, P = 9.3x10 − 5 ; Fig. 8 E). However, in the HER2-positive/ER-negative subtype, the relationship between HAVCR1 expression and patient response to chemotherapies appears to be opposite to that in the subtypes in which responders had significantly higher HAVCR-1 levels than nonresponders did (72 vs 37, P = 0.04; Fig. 8 D). 4.6.2 All-chemotherapy response on the basis of RFS at 5 years Using probe 207052_at, we examined whether HAVCR-1 transcript levels are associated with chemotherapy-induced RFS at 5 years across all MSs (responders, n = 256; nonresponders, n = 220) (Fig. 9 ). The responders presented markedly lower median expression than did the nonresponders (median 58 vs 96; P = 1.5 × 10⁻⁶, Fig. 9 A). Subgroup analysis also revealed that patients who responded to chemotherapies had significantly lower median HAVCR-1 expression than nonresponders in the luminal A subtype (median 50 vs 88, P = 0.014; Fig. 9 B), luminal B subtype (median 109 vs 138, P = 0.0047; Fig. 9 C) and TNBC subtype (median 46 vs 74, P = 0.014; Fig. 9 E). The HER2-positive/ER-negative subtypes showed a similar trend, although the difference was not statistically significant (P = 0.15; Fig. 9 D). 5. Discussion Breast carcinoma remains a global health burden and the principal driver of female cancer deaths [ 3 ] , underscoring the urgent need for novel biomarkers and druggable targets. Originally identified as the entry receptor for the hepatitis-A picornavirus HAVCR-1 is now recognized as a ubiquitously expressed membrane glycoprotein present in virtually all human tissues [ 6 ] . In addition to its canonical role in liver infection, this molecule has been implicated in autoimmune disorders [ 16 ] , allergic inflammation [ 17 ] and, increasingly, neoplastic progression. Elevated HAVCR-1 transcription has been linked to metastatic dissemination and adverse prognosis across multiple tumor types, including renal, hepatic and cervical cancers, making it an attractive candidate for targeted oncology therapeutics [ 12 – 13 ] . A recent study revealed that HAVCR-1 expression aligns with in situ breast carcinoma (P = 0.020), indicating that agents directed against HAVCR1 could be especially beneficial for tumors dominated by preinvasive components [ 18 ] . In our study, HAVCR-1 expression in breast cancer tissues was detected via real-time quantitative PCR and immunohistochemistry. The qPCR results revealed that HAVCR-1 expression is higher in tumor tissue than in normal tissue, which is in accordance with the immunohistochemistry results. The immunohistochemistry results indicated that HAVCR-1 protein was more highly expressed in breast cancer tissue than in normal tissue. In conclusion, HAVCR-1 expression in breast cancer tissues was upregulated. Additionally, we examined the correlations between HAVCR-1 expression and clinicopathological features, including the Nottingham prognostic index, histological classification, TNM stage, and breast cancer subtypes. The expression level of HAVCR-1 in Grade 1 invasive ductal carcinoma patients was significantly greater than that in Grade 2 and Grade 3 invasive ductal carcinoma patients (P < 0.05). For patients who died of breast cancer, HAVCR-1 expression was significantly higher in ER-positive patients than in ER-negative patients (P < 0.05). However, there was no significant difference in the expression of HAVCR-1 between patients with disease-free survival and those with recurrence and metastasis, a pattern that has also been observed in different subtypes of breast cancer. However, the survival curves revealed that patients with low HAVCR-1 expression had better RFS and OS than those with high HAVCR-1 expression. This finding was consistent with the HER2-positive subtype. Similarly, in the ER-positive subtype, low HAVCR-1 expression was associated with better OS. In contrast, in TNBC, HAVCR-1 expression was not correlated with prognosis. Overall, HAVCR-1 may serve as a potential prognostic marker for breast cancer. Furthermore, we explored the potential role of HAVCR-1 expression in influencing chemotherapeutic resistance or response. In combination with paclitaxel, docetaxel, a member of the taxane family, locks microtubules and remains a backbone of breast cancer therapy [ 19 ] . Our study revealed that the IC 50 values for WT and HAVCR-1 KD were 0.86 nM and 1.75 nM, respectively, for MCF-7 cells and 1.35 nM and 15.48 nM, respectively, for MDA-MB-231 cells, which indicates that reducing HAVCR-1 expression could enhance the resistance of breast cancer cells to docetaxel. This acquired resistance exemplifies the multifactorial hurdle that clinicians face, involving enhanced DNA repair, epithelial-to-mesenchymal shift, and evasion of apoptosis [ 20 ] . Finally, we explored a public platform, ROC plotter, which provides clinical information on patients’ response to chemotherapies. Notably, in patients who received chemotherapy in clinical practice, responders consistently presented lower HAVCR-1 transcript levels than nonresponders did, implying that diminished receptor expression may increase chemosensitivity, increase tumor regression and translate into a more favorable long-term prognosis. However, how low HAVCR-1 influences drug sensitivity in tumors remains mechanistically elusive and warrants further investigation. 6. Conclusions The present study indicates that HAVCR-1 may be a prognostic biomarker for breast cancer. It is also a powerful indicator of a patient’s response to chemotherapies. Further research needs to be done to explore the underlying mechanisms involved. Abbreviations HAVCR1 Hepatitis A virus cellular receptor 1 TIM-1 T-cell immunoglobulin mucin-1 KIM-1 human kidney injury molecule-1 IHC immunohistochemistry TNBC triple-negative breast cancer RFS relapse-free survival OS overall survival Declarations Ethics approval and consent to participate The project followed the Declaration of Helsinki and its later revisions. The study was approved by the local ethics committee (Bro Taf Local Research Ethics Committee (Panel B) for the Bro Taf Health Board, Cardiff, UK, issued 10/12/2001, reference 01/4303), and informed consent was obtained from all individual participants. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding No funding. Authors' contributions Xiaoshan Cao: Formal analysis, Investigation, Methodology, Writing – original draft. Binbin Cong: Methodology, Software, Validation, Formal analysis. Wenguo Jiang: Conceptualization, Resources, Supervision, Project administration, Formal analysis. Hong Liu: Conceptualization, Supervision, Validation, Writing – review & editing. Tracey A. Martin: Conceptualization, Supervision, Validation, Writing – review & editing. Acknowledgements We would like to thank Amber Xinyu Li, Jimmy Jianyuan Zeng, Wenxiao Ji, and Xinguo Zhuang for their patient guidance in experimental techniques and data analysis. References Novikava N, Redjdal A, Bouaud J, et al. Clinical Decision Support Systems Applied to the Management of Breast Cancer Patients: A Scoping Review. Stud Health Technol Inf. 2023;29:305:353–6. Arnold M, Morgan E, Rumgay H, et al. Current and future burden of breast cancer: Global statistics for 2020 and 2040. Breast. 2022;66:15–23. Bray F, Laversanne M, Sung H, Soerjomataram I, Jemal A, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. Gu Y, Bui T, Muller WJ. Exploiting Mouse Models to Recapitulate Clinical Tumor Dormancy and Recurrence in Breast Cancer. Endocrinology. 2022;163(6):bqac055. Park M, Kim D, Ko S, et al. Breast Cancer Metastasis: Mechanisms and Therapeutic Implications. 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12:34:28","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83612,"visible":true,"origin":"","legend":"","description":"","filename":"d572639bf0a846a388aa67108f67140e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/88bfce7ba7ce5db1a4c9646c.xml"},{"id":97897360,"identity":"c360a082-9fb6-448a-bdff-026846631ede","added_by":"auto","created_at":"2025-12-10 15:37:46","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95304,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/0ba2ce990ee061ad55642b47.html"},{"id":97895963,"identity":"d91062b2-ed2f-4314-9078-126792f06687","added_by":"auto","created_at":"2025-12-10 15:35:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":601278,"visible":true,"origin":"","legend":"\u003cp\u003eTranscript expression profile of HAVCR-1 in ductal carcinoma in comparison with grade information of the Cardiff breast cancer cohort. Two-sided t test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/296ba9578909b0610084d9be.png"},{"id":97897461,"identity":"7bad1fac-eba5-4495-b0ca-78241708b19c","added_by":"auto","created_at":"2025-12-10 15:37:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":747933,"visible":true,"origin":"","legend":"\u003cp\u003eTranscript expression of HAVCR-1 in different subtypes of the Cardiff breast cancer cohort. Sv4: Patients who died of breast cancer; sv234: Patients who have breast cancer-related incidence\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/76b598b1bbe26253124ae916.png"},{"id":97896030,"identity":"69991af0-33f0-4ccf-8f3e-15a97ea2a2e6","added_by":"auto","created_at":"2025-12-10 15:35:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506955,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of human breast samples. The arrows show positive staining forHAVCR-1.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/538951c945fabf0b96ce9d93.jpeg"},{"id":97794302,"identity":"c1fe2f01-20fe-4aa4-973c-2a915ce25e75","added_by":"auto","created_at":"2025-12-09 12:34:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1192453,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve showing the impact of HAVCR-1 expression on patient relapse-free survival (RFS) in the Cardiff clinical cohort. (A) Patients with low HAVCR-1 levels had longer RFS than those with high HAVCR-1 levels did (P=0.024). (B) Patients with low HAVCR-1 levels in the ER-positive subtype were found to have better RFS than those with high HAVCR-1 levels, but the difference was not statistically significant (P=0.059). (C) Patients with low levels of HAVCR-1 in the HER-2-positive subtype were found to have significantly better RFS than those with high levels of HAVCR-1 (P=0.003). (D) Patients with low HAVCR-1 levels in the TNBC subtype were found to have better RFS than those with high HAVCR-1 levels in theTNBC subtype, but the difference was not statistically significant (P=0.346).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/ba322ee6462bbb42e506de9a.png"},{"id":97794306,"identity":"0ecfa903-21fb-4f54-95b7-e1849ee6f320","added_by":"auto","created_at":"2025-12-09 12:34:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1038641,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve showing the impact of HAVCR-1 expression on overall survival in patients in the Cardiff clinical cohort. (A) Patients with low HAVCR-1 levels had longer OS than did those with high HAVCR-1 levels (P\u0026lt;0.001). (B) Patients with low levels of HAVCR-1 in the ER-positive subtype had worse OS than those with high levels of Her2 (P=0.038). (C) Patients with low levels of HAVCR-1 in the HER-2-positive subtype were found to have worse OS than those with high levels of HER-2 (P=0.002). (D) Patients with low HAVCR-1 levels in the TNBC subtype were found to have worse OS than those with high HAVCR-1 levels in theTNBC subtype (P=0.494).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/f3f4d54be55d98eacdd3ae9c.png"},{"id":97794315,"identity":"1d8fdeca-be2d-4cc7-9a3b-5061dfcf2b02","added_by":"auto","created_at":"2025-12-09 12:34:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":269660,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of HAVCR1 knockdown in MDA-MB-231 (A) and MCF-7 (B) cells by qPCR. *, P\u0026lt;0.05; ****, P\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/d9f7c2bab07f1b66ff14faad.png"},{"id":97896021,"identity":"98b4a4c8-141e-446e-aa65-54da30cf1514","added_by":"auto","created_at":"2025-12-10 15:35:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":718729,"visible":true,"origin":"","legend":"\u003cp\u003eCell toxicity of MDA-MB-231 and MCF-7 (WT vs. KD) cells treated with different concentrations of docetaxel.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/ce113c766d988fe7ba22a224.png"},{"id":97897747,"identity":"0291bc35-5323-4bf5-94e2-bdf8b78ba265","added_by":"auto","created_at":"2025-12-10 15:38:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1708551,"visible":true,"origin":"","legend":"\u003cp\u003eImplication of HAVCR-1 for the response to any neoadjuvant chemotherapy as determined by the pathological response from the ROC plotter. A. HAVCR-1 (207052_s_at) was detected in all subtypes. Nonresponders: n=1100;responders: n=532. B. HAVCR-1 (207052_s_at) was detected in the luminal A subtype. Nonresponders: n=341;responders: n=134. C. HAVCR-1 (207052_s_at) was detected in the luminal B subtype. Nonresponders: n=372;responders: n=119. D. HAVCR-1 (207052_s_at) was detected in the HER2-positive ER-negative subtype. Nonresponders: n=110; responders: n=83. E. HAVCR-1 (207052_s_at) was detected in the TNBC subtype. Nonresponders: n=277;responders: n=196. Raw data, box plots and ROC curves were analyzed and obtained from ROC plotter (http://www.rocplot.org/, accessed in March 2024). The Mann‒Whitney test was applied. Whiskers represent the maximum and minimum values. Mann‒Whitney test p value.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/902879f75bd0744269434777.png"},{"id":97794301,"identity":"dd64028d-52a6-48cf-bf06-15b8096632f7","added_by":"auto","created_at":"2025-12-09 12:34:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1671622,"visible":true,"origin":"","legend":"\u003cp\u003eROC plot of the effect of HAVCR-1 on resistance to any type of chemotherapy in advanced breast cancer patients (www.rocplot.org). A. HAVCR-1 (207052_s_at) was detected in all subtypes. Nonresponders: n=220; responders: n=256. B. HAVCR-1 (207052_s_at) was detected in the luminal A subtype. Nonresponders: n=20; responders: n=58. C. HAVCR-1 (207052_s_at) was detected in the luminal B subtype. Nonresponders: n=90;responders: n=83. D. HAVCR-1 (207052_s_at) was detected in the HER2-positive ER-negative subtype. Nonresponders: n=26, responders: n=35. E. HAVCR-1 (207052_s_at) was detected in the TNBC subtype. Nonresponders: n=84;responders: n=80. Raw data, box plots and ROC curves were analyzed and obtained from ROC plotter (http://www.rocplot.org/, accessed in March 2024). The Mann‒Whitney test was applied. Whiskers represent the maximum and minimum values.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/d1b251a580516c5f84ae3e87.png"},{"id":97903263,"identity":"a0d48090-7161-4c08-b9a6-f67f1b6f0f52","added_by":"auto","created_at":"2025-12-10 15:54:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9110477,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7939575/v1/0b572184-1466-427b-99ff-1791c9df254b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical Significance of Hepatitis A Virus Cellular Receptor 1 (HAVCR-1) in Breast Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCurrently, the incidence of breast carcinoma is increasing worldwide, and its impact continues to widen \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. More than 2.3\u0026nbsp;million fresh diagnoses and approximately 685 000 deaths were recorded in 2020, and demographic aging alone is expected to increase these numbers beyond 3\u0026nbsp;million cases and 1\u0026nbsp;million deaths annually by 2040 \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Breast cancer continues to have a large impact on the number of cancer deaths worldwide \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Once tumor cells disseminate to distant organs, the disease becomes largely incurable \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e; hence, there is an urgent need for fresh molecular flags and druggable targets that can anticipate or blunt progression.\u003c/p\u003e\u003cp\u003eThe hepatitis A virus entry factor HAVCR-1\u0026mdash;also annotated as T-cell immunoglobulin mucin-1 (TIM-1) or human kidney injury molecule-1 (KIM-1)\u0026mdash;was first identified as the portal for hepatotropic picornaviruses, yet its transcriptional territory extends far beyond the liver \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Robust baseline expression has been documented in the kidney, testis, small bowel, colon and spleen, implying broader physiological roles \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Accumulating evidence links HAVCR-1 to aggressive solid tumors: clear-cell renal carcinoma \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, cervical cancer \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, gastric adenocarcinoma \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and colorectal cancer \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e all display gain-of-expression signatures that correlate with adverse stages and outcomes, positioning the receptor as a candidate oncogenic driver.\u003c/p\u003e\u003cp\u003eFurthermore, functional assays have shown that HAVCR-1 promotes cell motility, matrix invasion and anchorage-independent growth \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. while its presence on activated T and NK cells modulates cytokine release and immune surveillance\u0026mdash;properties that could sculpt the tumor microenvironment \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, to the best of our knowledge, the expression of HAVCR-1 and its precise biological function in breast cancer are ambiguous. On the basis of these observations and previous studies, we hypothesized that HAVCR-1 could play a role in the occurrence and development of breast cancer. To verify this hypothesis, we evaluated the expression of HAVCR-1 in breast cancer and the relationship between HAVCR-1 expression and the prognosis of patients with HAVCR-1.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Breast cancer tissue collection\u003c/h2\u003e\u003cp\u003eA total of 127 breast cancer specimens and 33 adjacent histologically normal tissues were harvested intraoperatively, snap-frozen in liquid nitrogen, and stored until analysis; all donors provided written informed consent. The study protocol was approved by the Bro Taf Local Research Ethics Committee. Clinicopathological data were extracted from reports verified by a specialist pathologist, and postoperative surveillance was maintained for a median duration of 120 months.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 RNA extraction, reverse transcription and quantitative polymerase chain reaction (PCR)\u003c/h2\u003e\u003cp\u003eThe breast tissue pieces and cell line pellets were homogenized in AB Gene Total RNA reagent (Advanced Biotechnologies, UK). The RNA yield and 260/280 purity ratios were recorded on a WPA UV-1101 spectrophotometer (Biotech Photometer, UK). First-strand cDNA was synthesized from 0.25 \u0026micro;g of RNA with a Sigma reverse-transcription kit (Poole, UK) and checked with podoplanin (PDPLN) primers.\u003c/p\u003e\u003cp\u003eHAVCR-1 transcript numbers were quantified with the Amplifluor UniPrimer system (InterGen, UK) on an iCycler IQ thermal cycler (Bio-Rad, USA). The reactions contained Abgene qPCR MasterMix and were run at 95\u0026deg;C for 15 min, followed by 65 cycles at 95\u0026deg;C for 15 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 20 s. A plasmid dilution series generated copy number standards; β-actin served as the endogenous control, and cytokeratin-19 (CK19) was used to correct for the epithelial content. The primer/probe sequences are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; detailed protocols were reported previously \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers and probes for real-time quantitative PCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003emolecule\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward Primers (5\u0026rsquo;\u0026rarr;3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse Primers (5\u0026rsquo;\u0026rarr;3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTGAGTACGTCGTGGAGTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eACTGAACCTGACCGTACA\u003c/span\u003eCAGAGATGATGACCCTTTTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHAVCR-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACAATGACTGTTTCAACGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eACTGAACCTGACCGTACA\u003c/span\u003eTGGAGGAACAAAGGTAGAGA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytokeratin 19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAGGTCCGAGGTTACTGAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eACTGAACCTGACCGTACA\u003c/span\u003eCACTTTCTGCCAGTGTGTCTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: The Z sequence is highlighted in bold.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Immunohistochemical staining of breast samples\u003c/h2\u003e\u003cp\u003eSix-micron cryosections were prepared from snap-frozen tumors and matched background tissue (Leica DMB cryostat, Milton Keynes, UK), collected on SuperFrost Plus slides, and fixed in ice-cold acetone for 15 min. After brief air-drying and rehydration, nonspecific binding was blocked with 0.6% bovine serum albumin for 20 min. The sections were incubated for 1 h with HAVCR-1 primary antibody (70R-14161) or with antibody diluent alone (negative control), followed by three Tris-buffered saline washes. Biotinylated secondary antibody was applied for 30 min, the signal was amplified with VECTASTAIN ABC reagent (Vector Laboratories, USA), and the color was developed with diaminobenzidine (5 min, dark). The slides were counterstained with Gill\u0026rsquo;s hematoxylin, dehydrated through graded methanol, cleared in xylene, and coverslipped. Immunoreactivity was evaluated independently by two investigators.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell culture\u003c/h2\u003e\u003cp\u003eMCF-7 and MDA-MB-231 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM; Sigma, Poole, UK) supplemented with 10% fetal bovine serum and 1% penicillin‒streptomycin at 37\u0026deg;C in a humidified 5% CO₂ atmosphere.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 HAVCR-1 knockdown (KD)\u003c/h2\u003e\u003cp\u003eHAVRC-1 was knocked down in the MCF-7 and MDA-MB-231 cell lines through siRNA (sc-61691 Santa Cruz Biotechnology Inc., USA) transfection. For optimal transfection efficiency, Santa Cruz Biotechnology\u0026rsquo;s siRNA transfection reagent (sc-29528), siRNA transfection medium (sc-36868) and siRNA dilution buffer (sc-29527) are recommended.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Docetaxel cytotoxicity assays\u003c/h2\u003e\u003cp\u003eTo test whether HAVCR-1 modulates docetaxel tolerance, wild-type and HAVCR-1-silenced MCF-7 and MDA-MB-231 cells were plated at 5 \u0026times; 10\u0026sup3; per well (96-well format) in 100 \u0026micro;L of medium containing a 2-log docetaxel gradient (MCF-7: 0.02\u0026ndash;2 000 nM; MDA-MB-231: 0.48\u0026ndash;48 000 nM) or drug-free control (six replicates). After 72 h at 37\u0026deg;C/5% CO₂, the medium was removed, the cells were gently rinsed with PBS, fixed with 4% paraformaldehyde (10 min, room temperature), and stained with 0.5% crystal violet (10 min). After these plates were dried, the dye was solubilized in 10% (v/v) acetic acid (100 \u0026micro;L/well), and the absorbance was read at 595 nm on an LT-4500 microplate reader. Survival curves were constructed in Microsoft Excel.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Statistical analysis","content":"\u003cp\u003eTranscript levels of HAVCR-1 between two groups were compared with two-tailed Student\u0026rsquo;s t test under normality or the Mann\u0026ndash;Whitney U test when normality was rejected. For \u0026gt;\u0026thinsp;2 groups, one-way ANOVA plus Tukey\u0026rsquo;s post hoc test was applied to normally distributed data; otherwise, the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s correction was used. Association strength and predictive value were examined via χ\u0026sup2; tests and receiver operating characteristic (ROC) curve analysis in SPSS 27 (IBM UK, Porto). Plots were generated with GraphPad Prism 9.5 (San Diego, CA). A two-sided P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant and is indicated as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Expression of HAVCR-1 in breast cancer\u003c/h2\u003e\u003cp\u003eHAVCR-1 mRNA was quantified by qPCR in a Cardiff cohort of breast carcinomas paired with adjacent normal tissue. Although median levels were elevated in tumors, the change did not reach statistical significance (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and no associations emerged with standard clinicopathological variables.\u003c/p\u003e\u003cp\u003eWhen ductal carcinomas were stratified by grade, Grade 1 cases presented markedly higher HAVCR-1 transcript counts than Grade 2 or Grade 3 lesions did (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among patients who ultimately died from breast cancer, those with ER-positive tumors presented significantly greater HAVCR-1 expression than those with ER-negative primaries did (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e); in contrast, no difference was observed between those who remained disease-free and those who developed progression (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTranscript expression profile of HAVCR-1 in comparison with the clinicopathological information of the Cardiff breast cancer cohort. Kruskal‒Wallis ANOVA on RANKS.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSample number (n)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMedian transcript expression\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eQ1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eQ3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNormal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.2697\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTumor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e316\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eNottingham prognostic index\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;=\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.105\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2.4-\u0026lt;=3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1319\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eTumor Grade\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8316\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.098\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e173\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e228\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eTNM stage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.894\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e365\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eClinical outcome\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease free\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e379\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.253\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWith Metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e429\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWith local recurrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e544\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDied of breast cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1093\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eNottingham prognostic index in ductal breast cancer\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;=\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.316\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;2.4-\u0026lt;=3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e877\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e221\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eTNM in ductal breast cancer\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e702\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.817\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e330\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eClinical outcome in ductal breast cancer\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDisease free\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.291\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWith Metastasis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e429\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWith local recurrence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e365\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDied of breast cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1348\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: a Mann‒Whitney test; b Kruskal‒Wallis ANOVA on RANKS\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Immunohistochemical staining of HAVCR-1 expression in clinical breast cancer tissue\u003c/h2\u003e\u003cp\u003eHistorical fresh frozen breast tissue sections from a set of samples previously available in our laboratory were utilized to explore HAVCR-1 protein expression levels. Representative micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) show consistently denser tumor-associated immunostaining relative to adjacent normal parenchyma.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Impact of HAVCR-1 expression on relapse-free survival and overall survival in breast cancer patients\u003c/h2\u003e\u003cp\u003eTo evaluate the prognostic value of HAVCR-1, we constructed relapse-free survival (RFS) and overall survival (OS) curves for the Cardiff series using the cohort-specific median transcript level as the cutoff. Patients below the median (low HAVCR-1, n\u0026thinsp;=\u0026thinsp;85) had significantly better RFS (mean\u0026thinsp;=\u0026thinsp;125.0 months) than those with high HAVCR-1 (n\u0026thinsp;=\u0026thinsp;13) (mean\u0026thinsp;=\u0026thinsp;87.8 months) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Analysis of the effect of HAVCR-1 expression on the HER-2-positive subtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) revealed that patients who expressed low levels of HAVCR-1 (n\u0026thinsp;=\u0026thinsp;41) had significantly better RFS than those who expressed high levels of HAVCR-1 (P\u0026thinsp;=\u0026thinsp;0.003). The trends in the ER-positive subtype and TNBC subtype were similar to those in the HER2 subtype, without statistical significance (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe relationship between HAVCR-1 expression and patient OS was also explored in the Cardiff cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Patients who presented with low HAVCR-1 levels (n\u0026thinsp;=\u0026thinsp;85) had significantly better OS (mean\u0026thinsp;=\u0026thinsp;141.5 months) than did those with high HAVCR-1 levels (n\u0026thinsp;=\u0026thinsp;13) (mean\u0026thinsp;=\u0026thinsp;91.5 months) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the subgroup analysis, high HAVCR-1 expression was associated with worse OS in the ER-positive (P\u0026thinsp;=\u0026thinsp;0.038, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and HER-2-positive subtypes (P\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Expression and knockdown of HAVCR-1\u003c/h2\u003e\u003cp\u003eHAVCR-1 levels were significantly lower in HAVCR-1-KD MDA-MB-231 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and MCF-7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) cells than in WT cells at the mRNA level, confirming that HAVCR-1-KD cell models were successfully established with breast cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.5 HAVCR-1 KD enhances breast cancer cell resistance to docetaxel\u003c/h2\u003e\u003cp\u003eFollowing the incubation of the cellular models in serially diluted docetaxel for 72 hours, the loss of HAVCR-1 rendered both MCF-7 and MDA-MB-231 cells resistant to docetaxel (the IC50 values for WT and HAVCR1 KD were 0.86 nM and 1.75 nM for MCF-7 cells and 1.35 nM and 15.48 nM for MDA-MB-231 cells, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.6 HAVCR-1 as a predictor of chemotherapy response in the breast cancer ROC plotter cohort\u003c/h2\u003e\u003cp\u003eTo examine whether HAVCR-1 modulates resistance to systemic therapy, we mined the ROC plotter resource (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rocplot.org\u003c/span\u003e\u003cspan address=\"http://www.rocplot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a compendium of 3 104 breast cancer transcriptomes with annotated drug-response calls \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The response was defined according to the Response Evaluation Criteria in Solid Tumors 1.1 criteria. Transcript levels between responders and nonresponders were visualized as box-and-whisker plots and compared with the Mann\u0026ndash;Whitney U test; ROC curves were generated to quantify the predictive accuracy of HAVCR-1 for both chemotherapy and targeted agents (Fekete \u0026amp; Győrffy, 2019).\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e4.6.1 All-chemotherapy response on the basis of pathological complete response\u003c/h2\u003e\u003cp\u003eProbe set 207052_at was used to compare HAVCR-1 transcript levels with pathological response after any neoadjuvant chemotherapy across all ROC-plotter subtypes (responders, n\u0026thinsp;=\u0026thinsp;532; nonresponders, n\u0026thinsp;=\u0026thinsp;1100) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, patients who responded to chemotherapies had significantly lower median HAVCR-1 expression than nonresponders did (median 36 vs 90, P\u0026thinsp;=\u0026thinsp;8.7x10\u003csup\u003e\u0026minus;\u0026minus;18\u003c/sup\u003e). The beneficial effects of low-level HAVCR1 and patient sensitivity to chemotherapies were observed in the luminal A subtype (5 vs 74, P\u0026thinsp;=\u0026thinsp;3.7x10\u003csup\u003e\u0026minus;\u0026thinsp;19\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), luminal B subtype (median 112 vs 139, P\u0026thinsp;=\u0026thinsp;0.00062; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) and TNBC subtype (median 32 vs 63, P\u0026thinsp;=\u0026thinsp;9.3x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). However, in the HER2-positive/ER-negative subtype, the relationship between HAVCR1 expression and patient response to chemotherapies appears to be opposite to that in the subtypes in which responders had significantly higher HAVCR-1 levels than nonresponders did (72 vs 37, P\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e4.6.2 All-chemotherapy response on the basis of RFS at 5 years\u003c/h2\u003e\u003cp\u003eUsing probe 207052_at, we examined whether HAVCR-1 transcript levels are associated with chemotherapy-induced RFS at 5 years across all MSs (responders, n\u0026thinsp;=\u0026thinsp;256; nonresponders, n\u0026thinsp;=\u0026thinsp;220) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The responders presented markedly lower median expression than did the nonresponders (median 58 vs 96; P\u0026thinsp;=\u0026thinsp;1.5 \u0026times; 10⁻⁶, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Subgroup analysis also revealed that patients who responded to chemotherapies had significantly lower median HAVCR-1 expression than nonresponders in the luminal A subtype (median 50 vs 88, P\u0026thinsp;=\u0026thinsp;0.014; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), luminal B subtype (median 109 vs 138, P\u0026thinsp;=\u0026thinsp;0.0047; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC) and TNBC subtype (median 46 vs 74, P\u0026thinsp;=\u0026thinsp;0.014; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). The HER2-positive/ER-negative subtypes showed a similar trend, although the difference was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.15; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eBreast carcinoma remains a global health burden and the principal driver of female cancer deaths \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, underscoring the urgent need for novel biomarkers and druggable targets. Originally identified as the entry receptor for the hepatitis-A picornavirus HAVCR-1 is now recognized as a ubiquitously expressed membrane glycoprotein present in virtually all human tissues \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In addition to its canonical role in liver infection, this molecule has been implicated in autoimmune disorders \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, allergic inflammation \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and, increasingly, neoplastic progression. Elevated HAVCR-1 transcription has been linked to metastatic dissemination and adverse prognosis across multiple tumor types, including renal, hepatic and cervical cancers, making it an attractive candidate for targeted oncology therapeutics \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA recent study revealed that HAVCR-1 expression aligns with in situ breast carcinoma (P\u0026thinsp;=\u0026thinsp;0.020), indicating that agents directed against HAVCR1 could be especially beneficial for tumors dominated by preinvasive components \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In our study, HAVCR-1 expression in breast cancer tissues was detected via real-time quantitative PCR and immunohistochemistry. The qPCR results revealed that HAVCR-1 expression is higher in tumor tissue than in normal tissue, which is in accordance with the immunohistochemistry results. The immunohistochemistry results indicated that HAVCR-1 protein was more highly expressed in breast cancer tissue than in normal tissue. In conclusion, HAVCR-1 expression in breast cancer tissues was upregulated.\u003c/p\u003e\u003cp\u003eAdditionally, we examined the correlations between HAVCR-1 expression and clinicopathological features, including the Nottingham prognostic index, histological classification, TNM stage, and breast cancer subtypes. The expression level of HAVCR-1 in Grade 1 invasive ductal carcinoma patients was significantly greater than that in Grade 2 and Grade 3 invasive ductal carcinoma patients (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For patients who died of breast cancer, HAVCR-1 expression was significantly higher in ER-positive patients than in ER-negative patients (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, there was no significant difference in the expression of HAVCR-1 between patients with disease-free survival and those with recurrence and metastasis, a pattern that has also been observed in different subtypes of breast cancer. However, the survival curves revealed that patients with low HAVCR-1 expression had better RFS and OS than those with high HAVCR-1 expression. This finding was consistent with the HER2-positive subtype. Similarly, in the ER-positive subtype, low HAVCR-1 expression was associated with better OS. In contrast, in TNBC, HAVCR-1 expression was not correlated with prognosis. Overall, HAVCR-1 may serve as a potential prognostic marker for breast cancer.\u003c/p\u003e\u003cp\u003eFurthermore, we explored the potential role of HAVCR-1 expression in influencing chemotherapeutic resistance or response. In combination with paclitaxel, docetaxel, a member of the taxane family, locks microtubules and remains a backbone of breast cancer therapy \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Our study revealed that the IC\u003csub\u003e50\u003c/sub\u003e values for WT and HAVCR-1 KD were 0.86 nM and 1.75 nM, respectively, for MCF-7 cells and 1.35 nM and 15.48 nM, respectively, for MDA-MB-231 cells, which indicates that reducing HAVCR-1 expression could enhance the resistance of breast cancer cells to docetaxel. This acquired resistance exemplifies the multifactorial hurdle that clinicians face, involving enhanced DNA repair, epithelial-to-mesenchymal shift, and evasion of apoptosis \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFinally, we explored a public platform, ROC plotter, which provides clinical information on patients\u0026rsquo; response to chemotherapies. Notably, in patients who received chemotherapy in clinical practice, responders consistently presented lower HAVCR-1 transcript levels than nonresponders did, implying that diminished receptor expression may increase chemosensitivity, increase tumor regression and translate into a more favorable long-term prognosis. However, how low HAVCR-1 influences drug sensitivity in tumors remains mechanistically elusive and warrants further investigation.\u003c/p\u003e"},{"header":"6. Conclusions","content":"\u003cp\u003eThe present study indicates that HAVCR-1 may be a prognostic biomarker for breast cancer. It is also a powerful indicator of a patient\u0026rsquo;s response to chemotherapies. Further research needs to be done to explore the underlying mechanisms involved.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHAVCR1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHepatitis A virus cellular receptor 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTIM-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eT-cell immunoglobulin mucin-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKIM-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehuman kidney injury molecule-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimmunohistochemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNBC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etriple-negative breast cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelapse-free survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eoverall survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project followed the Declaration of Helsinki and its later revisions. The study was approved by the local ethics committee (Bro Taf Local Research Ethics Committee (Panel B) for the Bro Taf Health Board, Cardiff, UK, issued 10/12/2001, reference 01/4303), and informed consent was obtained from all individual participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoshan Cao: Formal analysis, Investigation, Methodology, Writing \u0026ndash; original draft. Binbin Cong: Methodology, Software, Validation, Formal analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWenguo Jiang: Conceptualization, Resources, Supervision, Project administration, Formal analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHong Liu: Conceptualization, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing. Tracey A. Martin: Conceptualization, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Amber Xinyu Li, Jimmy Jianyuan Zeng, Wenxiao Ji, and Xinguo Zhuang for their patient guidance in experimental techniques and data analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNovikava N, Redjdal A, Bouaud J, et al. Clinical Decision Support Systems Applied to the Management of Breast Cancer Patients: A Scoping Review. 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The T-cell immunoglobulin and mucin domain (Tim) gene family in asthma, allergy, and autoimmunity. Allergy Asthma Proc. 2013;34(1):e21-6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDiniz G, Pulular AG, Solakoğlu Kahraman D, Varol U, Sayhan S, Ayaz D, Karaali C. Tissue Expression of Neutrophil Gelatinase-Associated Lipocalin and Kidney Injury Molecule-1 in Breast Cancers. Eur J Breast Health. 2022;18(4):336\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIsmail U, Killeen RB, Taxane T. 2023 Mar 12. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan\u0026amp;#8211.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhanyamraju PK. Drug resistance mechanisms in cancers: Execution of pro-survival strategies. J Biomed Res. 2024;38(2):95\u0026ndash;121.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HAVCR-1, breast cancer, overall survival, relapse-free survival, chemotherapy response","lastPublishedDoi":"10.21203/rs.3.rs-7939575/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7939575/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Hepatitis A virus cellular receptor 1 (HAVCR-1), first discovered as the entry factor for hepatitis A virus, has since been recognized as a tumor-associated antigen and is currently being intensively explored as a prognostic biomarker across multiple malignancies, including gastric and colorectal cancers. However, its contribution to breast cancer remains ambiguous. This study investigated the expression and clinical significance of HAVCR-1 in breast cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e HAVCR-1 expression was evaluated via quantitative real-time PCR (qPCR) and immunohistochemistry (IHC) in a cohort of fresh-frozen normal and breast cancer tissues. Expression levels were correlated with clinicopathological parameters, including the Nottingham prognostic index, tumor stage, histological subtype, hormone receptor status (ER, PR, HER2), and survival outcomes. The prognostic and predictive value of HAVCR-1 was assessed via chi-square tests and receiver operating characteristic (ROC) analysis. The median follow-up period was 120 months. In vitro, HAVCR-1 was knocked down via siRNA in the MCF-7 and MDA-MB-231 cell lines, and the response to docetaxel was evaluated on the basis of the IC50 values. Additionally, ROC plotter analysis was used to assess the association between HAVCR-1 expression and therapeutic response in breast cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAlthough qPCR revealed no statistically significant difference in HAVCR-1 mRNA levels between tumor and normal tissues (P=0.2697), IHC revealed increased protein expression in breast cancer tissues. Low HAVCR-1 expression was significantly associated with improved overall survival (OS, P \u0026lt; 0.01) and relapse-free survival (RFS, P=0.04). Subgroup analysis indicated that the survival benefit of low HAVCR-1 expression was particularly evident in ER-positive (P=0.046) and HER2-positive (P=0.004) subtypes but not in triple-negative breast cancer (TNBC). In vitro knockdown of HAVCR-1 conferred resistance to docetaxel in both MCF-7 (IC50: 0.86 nM vs. 1.75 nM) and MDA-MB-231 (IC50: 1.35 nM vs. 15.48 nM) cells. Clinically, patients with high HAVCR-1 expression are more likely to exhibit resistance to chemotherapy, particularly those with the luminal A, luminal B, and TNBC subtypes, but not those with HER2+/ER− tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eHAVCR-1 is an independent prognostic factor for OS and RFS in breast cancer patients and may serve as a predictive biomarker for chemotherapy response. Its combination with ER, PR, and HER2 status could enhance prognostic stratification and therapeutic decision-making.\u003c/p\u003e","manuscriptTitle":"Clinical Significance of Hepatitis A Virus Cellular Receptor 1 (HAVCR-1) in Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 12:34:22","doi":"10.21203/rs.3.rs-7939575/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-20T10:16:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46564140585925708403776520678907693717","date":"2026-05-20T04:47:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-25T20:47:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230640556656829005284194836238322352291","date":"2025-12-11T17:32:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315457073133271247949404416416367448827","date":"2025-12-06T03:43:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-05T14:59:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T18:43:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-11T10:59:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-11T09:38:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-11-11T09:33:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f54c51e9-5dcf-4e90-89f6-a528b342f851","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-20T10:16:57+00:00","index":92,"fulltext":""},{"type":"reviewerAgreed","content":"46564140585925708403776520678907693717","date":"2026-05-20T04:47:04+00:00","index":91,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-09T12:34:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-09 12:34:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7939575","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7939575","identity":"rs-7939575","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}