Prognostic role of HIST1H4C expression and neoadjuvant therapy efficacy in breast cancer

Prognostic role of HIST1H4C expression and neoadjuvant therapy efficacy 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 Prognostic role of HIST1H4C expression and neoadjuvant therapy efficacy in breast cancer Li Qian, Rui Ge, Zhu Cairong, Zhong Haihu, Cai Yuanxuan, Liao Yongdong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4525730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose breast cancer is a common and highly malignant, currently, HIST1H4C was found to be associated with several human malignancies. The purpose of this study is to investigate tissue HIST1H4C expression in breast cancer and explore its role in disease progression and its interaction with neoadjuvant therapy efficacy. Methods we analyzed tissue HIST1H4C mRNA expression in BC tissue samples from 105 patients received with neoadjuvant therapy using qPCR between 2019–2022. Results Statistical analysis showed that a high expression of HIST1H4C before neoadjuvant therapy was positively related to good responder (CR + PR), while high expression of HIST1H4C after neoadjuvant therapy was negatively related good responder. And HIST1H4C expression was significantly decreased in patients with good responder. In addition, high HIST1H4C expression was also related to ER negative, PR negative, high KI67 expression, high level of histological grade, large tumor size and more lymph node metastases in Curtis database. Furthermore, high HIST1H4C expression before and after-treatment in our center or in database has a positively correlation with poor prognosis. Conclusions HIST1H4C is the potential biomarker of neoadjuvant therapy and prognosis for breast cancer. HIST1H4C neoadjuvant therapy efficacy prognosis BC Figures Figure 1 Figure 2 Figure 3 Introduction Breast cancer (BC) is the most common malignant tumor in women worldwide[ 1 ]. Chemotherapy plays a crucial role in the systemic treatment of breast cancer, with neoadjuvant chemotherapy (NAC) being particularly important. NAC aims to shrink tumor size before surgery, making inoperable breast cancer operable and improving surgical success rates. It also provides valuable information on drug sensitivity and guides subsequent treatment.[ 2 ] Studies have shown that NAC can improve prognosis, increase the rate of pathological complete response (pCR), and enhance event-free survival (EFS) in breast cancer patients.[ 3 – 5 ] In 2014, the CTNeoBC study validated that achieving pCR following NAC was associated with a more favorable prognosis.[ 6 ] The I-SPY2 study further confirmed that hormone receptor (HR) -positive breast cancer patients who achieved pCR had significantly prolonged survival.[ 7 ] However, a significant number of HR-positive breast cancer patients exhibit chemotherapy resistance, and NAC may lead to an inadequate tumor shrinkage or downstaging and unexpected and irreversible adverse reactions. Therefore, identifying patients who benefit most from NAC and tailoring personalized treatment strategies is crucial. Tumor heterogeneity is reflected in tumor pathology grade[ 8 ], various classification systems are used globally, with the Histological Classification System (HCS) being one of the most widely recognized. Histologically, the malignancy level of breast cancer is closely associated with its grade, which correlates with the proliferation index and DNA ploidy[ 9 ]. As a result, this index holds considerable significance in assessing the differentiation level and prognosis of breast cancer[ 9 , 10 ]. Invasive ductal carcinoma, in particular, exhibited significantly more responsiveness to chemotherapy compared to non-invasive ductal carcinoma, and treatment approaches targeting higher histological grades were notably more effective than those targeting lower grades. Studies demonstrated a positive correlation between histological grade and the pCR rate.[ 11 , 12 ] Therefore, histological grade serves as a crucial predictor of the response to NAC. Our previous research involved analyzing single-cell sequencing data sets from GSE161529 and GSE176078. We obtained 166298 and 84320 scRNA-seq data points from 58 breast cancer samples after filtrating by nCount_RNA > 1000 and nCount_RNA < 10000 in 2 database above (Fig. S1 A-B). Epithelial cells primarily expressed EPCAM, KRT7, KRT8 and patients were divided into low-grade (I-II) and high-grade (III) histological grade groups for further analysis. Tumor cells were classified into 6 subgroups, with cluster-1 showing increased proportion in the high-grade histological grade subgroup (Fig. S1 C-F). Analysis revealed high expression of HIST1H4C in cluster-1 in both 2 dataset (Fig. S1 G-H), and the trajectory of differentiation in tumor cells predicted by monocle 3 showed cluster-1 may be the origin of tumor cells (Figure. S1I-J), indicating its importance in tumor development. This study aimed to investigate the prognostic role of HIST1H4C and its interaction with the efficacy of neoadjuvant therapy and prognosis in breast cancer patients. Methods mRNA extraction and Quantitative PCR with reverse transcription TRIzol® Reagent (Life Technologies, USA) was used to extract total RNA from tumor tissue. RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (RR036A, Takara). Real-time quantitative PCR was performed using TB Green Premix Ex Taq II (RR820A, Takara) according to the manufacturer’s recommendations. The reactions were carried out in the LightCycler480 system with gene-specific primers. Standard dilution series were utilized to estimate the efficiency of the hic assay. Threshold cycles (Ct) were employed to calculate mRNA expression compared to the respective housekeeping gene GAPDH by relative quantification using the 2exp −ΔΔCt method. Patients in roll The study included female patients diagnosed with stage II breast cancer who underwent neoadjuvant chemotherapy (NAC) at Guangzhou Women and Children's Medical Center from August 2019 to November 2022. Inclusion criteria were: women aged 18 or older who signed an informed consent form, non-metastatic breast cancer patients with TNM stage II and above, no prior chemotherapy, endocrine therapy, surgery, or radiotherapy, normal cardiac function, ECOG score ≤ 2, and good organ function. Exclusion criteria were: patients with other malignancies in the last 5 years, or not suitable for chemotherapy. The study was a retrospective observational clinical study with the primary endpoint being pathological complete response (pCR) after NAC. Secondary endpoints included breast preservation surgery rate and prognosis-free survival (PFS) and overall survival (OS). Data collection included diagnosis confirmation, imaging examinations, molecular typing, chemotherapy, and surgical outcomes evaluation. Discrepancies of case inclusion were conferred by a principal investigator. The flowchart describing patient screening is shown in Figure S2. Treatment protocol The NAC regimen was adriamycin (60 mg/m 2 ) + cyclophosphamide (600 mg/m 2 ) every 3 weeks for 4 cycles, followed by paclitaxel (80 mg/m 2 ) for 12 weeks or docetaxel (75 mg/m 3 ) and cyclophosphamide (600 mg/m 3 ) for four to six cycles every three weeks. HER2 + BC patients were treated with double-target therapy (trastuzumab plus pertuzumab). The therapeutic efficacy of the regimens was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST). pCR was defined as the absence of any residual invasive carcinoma or ductal carcinoma in situ (DCIS) in the breast or lymph node tissue removed after NAC and examined under a microscope. Breast- or lymph node-only pCR was defined as the absence of any residual invasive cancer or DCIS confined to the breast or armpit, as determined by examination under a microscope[ 13 ]. Partial response (PR) was defined as a reduction of at least 30% in the sum of the longest diameter of the target lesion found by clinical examination and imaging. Progressive disease (PD) was defined as an increase of at least 20% in the sum of the longest diameter of the target lesion found by clinical examination and imaging. Stable disease (SD) was defined as neither sufficient shrinkage to qualify for PR nor sufficient growth to qualify for PD. pCR, which refers only to the breast and lymph nodes, was assessed only in patients with clinically positive lymph nodes at the time of diagnosis. Patients with pCR, breast/lymph node-specific pCR, and PR were classified as having treatment-sensitive BC, and patients with SD and PD were classified as having treatment-resistant BC. Statistical analysis A completely randomized, balanced design was used for all experiments. All comparison groups have the similar variance. Wilcoxon matched-pairs signed rank test or the Mann-Whitney U-test were used to test the significance of differences in various molecular, cellular, and physiological parameters between the means or medians in treatment and control groups. A P value less than 0.05 was considered significant. Error bars in the experiments indicate standard deviation (SD) for a minimum of three independent experiments. Result Patients in roll In order to investigate the correlation between HIST1H4C expression and the efficacy of neoadjuvant therapy (NACT) in breast cancer, we conducted a prospective study involving patients with newly diagnosed who had not received NACT before. A total of 109 patients were recruited from the Thyroid and Breast Department, the Guangzhou Women and Children's Medical Center in China between 2019 and 2022. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Guangzhou Women and Children's Medical Center. Detailed clinicopathological information of all patients is provided in Table S1 . The age of the enrolled patients ranged from 34 to 66 years, with 62 being premenopausal, 38 postmenopausal, and 5 whose menopausal status could not be determined due to oophorectomy. The median follow-up duration for this study, up to the cut-off date of May 1st, 2024, ranged from 16 to 63 months. High expression of HIST1H4C before treatment related to good response to neoadjuvant therapy High HIST1H4C expression before treatment is associated with a poor response to neoadjuvant therapy. HIST1H4C expression levels were determined using qPCR, with the median fold change of mRNA expression/GAPDH before treatment being 0.0023. Patients were classified as having a better response (pathologic complete response (pCR) or partial response (PR)) or a poor response (stable disease (SD) or progressive disease (PD)) to NACT. Results showed significantly higher HIST1H4C expression in good responders compared to poor responders (p < 0.001). (Fig. 1 A). The preoperative tumor grade, expression of ER, PR, HER2, and ki67 were determined by two physicians and we gained all tumor size messages before treatment through MR or ultrasonography. Though we only obtained pathological grade data of partly patients, 33 were evaluated as high grade (3) and 16 people were low grade (1–2). Within the limited samples, we analyzed the relationship between HIST1H4C expression and pathology grade and found pathological grade revealed no significant association between HIST1H4C expression (p = 0.13) (Fig. 1 B). In addition, our data also indicated baseline HIST1H4C expression differed in stage (T2 VS T3) and nodal status (N0 VS N1-3), We found higher HIST1H4C expression was positively correlated with lymph node metastasis but not with tumor size before treatment. (Fig. 1 C-D). Furthermore, HIST1H4C expression was found to be significantly higher in triple-negative breast cancer (TNBC) samples compared to other subtypes. (Fig. 1 E-H) Despite previous findings linking increased Ki67 expression to chemotherapy sensitivity, our results did not show an association between HIST1H4C and Ki67 levels. (Fig. 1 I). Kaplan-Meier analysis demonstrated that patients with high HIST1H4C expression before treatment had a poorer PFS to compared to those with low expression ( p = 0.015). (Fig. 1 J) Finally, baseline HIST1H4C levels showed a trend towards correlation with overall survival ( p = 0.06). (Fig. 1 K). High expression of HIST1H4C after treatment related to poor response to neoadjuvant therapy To further screen HIST1H4C expression after-treatment, qPCR also be conducted by tumor tissue HIST1H4C after-treatment. The median fold change of mRNA expression/GAPDH after-treatment was 0.0007. Firstly, the result suggests that higher expression of HIST1H4C after-treatment significantly related to poor response to NACT which is opposite to before-treatment (Fig. 2 A). Next, we analyzed the relationship between HIST1H4C expression and pathological grade after treatment and found higher pathological grade revealed significantly association to higher HIST1H4C expression. (Fig. 2 B) And tumor size and lymph node status messages after treatment was evaluated pathologically, different conclusion in some of clinical data was drawn out, as higher HIST1H4C expression after-treatment was positively correlated to larger tumor size, but the relationship between HIST1H4C after-treatment and lymph node status was unclear. (Fig. 2 C-D) Moreover, in our cohort, a small subset exhibited changes in pathological subtypes after treatment. Due to the inability to evaluate the ER, PR, HER2 and Ki67 post-treatment for T0/is patients, we assumed that the pathological analysis after treatment remained consistent as pre-treatment. Analysis using the chi-square test revealed HIST1H4C expression was higher in PR-negative or HER2-negative patients. (Table S1 ) Furthermore, an unpaired rank-sum test demonstrated a significant increase in HIST1H4C expression in PR-negative patients, although no such difference was observed for HER2-negative patients compared to HER2-positive patients. Neither comparison method found association between HIST1H4C expression and ER expression. Subsequently, following NACT, no significant difference in HIST1H4C expression was found among these subtypes in the postoperative specimens. (Fig. 2 E-H) Similarly, the relationship between post-treatment HIST1H4C expression and Ki67 level remains inconclusive (Fig. 2 I). Importantly, our findings suggest that high post-treatment HIST1H4C expression is associated with a poorer PFS ( p = 0.003). And finally, high post-treatment HIST1H4C showed a trend towards correlation with overall survival ( p = 0.11). (Fig. 2 J-K). The data above indicates that both baseline and post-NACT HIST1H4C could be important indicators in predicting the efficacy of NACT. HIST1H4C may be regulated by NACT, and its expression could potentially predict clinical prognosis. Decimated HIST1H4C correlated with good response to neoadjuvant therapy To investigate whether HIST1H4C may be regulated by NACT, a comparison between baseline and post-treatment was conducted. Paired t-tests revealed a significant downregulation of HIST1H4C by NACT. Patients who responded well showed a notable decrease in HIST1H4C expression, while patients with a poor response did not exhibit a significant decrease (Fig. 3 A-C). An effective decrease in HIST1H4C was defined as a reduction of over 50% as Decrease group, while a reduction of less than 50% or an increase was categorized as the Non-decrease group. Subsequent analysis showed that patients with lower grade levels (2), earlier stages (T2), less lymphatic metastasis (N0), or higher Ki67 expression did not demonstrate a more pronounced decimation of HIST1H4C (Fig. 3 D-G). Unfortunately, the data did not show a significant difference in Kaplan-Meier analysis between the decrease group and non-decrease group, indicating that patients with a significant reduction in HIST1H4C did not have improved progression-free survival (PFS) or overall survival (OS) (Fig. 3 H-I). High expression of HIST1H4C related to poor prognosis in CURTIS To determine the relationship between HIST1H4C expression and clinical data such as pathological grade and clinical prognosis, medical records were obtained from the CURTIS database. Analysis revealed a significant increase in HIST1H4C expression in high-grade (3) compared to low-grade (1–2) subgroups. (Fig.S3A) Additionally, the expression of HIST1H4C was found to be significantly associated with tumor size and lymph node metastasis in patients with higher levels of expression. Subpopulations in the CURTIS database was further divided based on CLAUDIN_SUBTYPE, including luminal A, luminal B, HER2+, TNBC. Comparison of HIST1H4C expression in triple-negative breast cancer (basal, normal, or claudin-low) indicated increased expression compared to luminal and HER2 + subsets, with no significant difference observed in the latter two subgroups. Moreover, higher expression of HIST1H4C was observed in ER-negative, PR-negative patients, with no significant difference in HIST1H4C expression seen in the HER2-negative and positive subgroups. (Fig.S3B and Table S2) Subsequent analysis by dividing patients into two groups based on median HIST1H4C levels revealed significantly shorter survival times in patients with high HIST1H4C expression, highlighting the potential prognostic value of HIST1H4C expression. (Fig.S3C) Discussion Breast cancer is the most common malignancy globally and a leading cause of cancer-related deaths in women[ 14 ]. Some breast cancers present with large tumor and metastatic lymph nodes at diagnosis, leading to a low 5-year survival rate. In the pursuit of optimal care, clinicians are striving to identify areas where 'downgrade' treatment can be effective[ 15 , 16 ]. pCR has long been associated with a better prognosis in patients undergoing NAC[ 6 , 17 ]. Although numbers potential benefits of NACT, the risks of delaying surgery with poor treatment response could develop unresectable disease and increased chances for metastatic tumor spread. In addition, the toxic effects of NACT might aggravate morbidity or further delay surgical resection, and increasing surgical risk.[ 18 , 19 ] Thereby, it is greatly need to develop a specific biomarker signature to define the ideal patient population for NAC. Various factors such as molecular typing, Ki67 expression, histological grade, tumor size, lymph node metastasis, and immune cell infiltration have been linked to the efficacy of NACT in breast cancer.[ 12 , 20 – 23 ] However, in the era of personalized therapy, additional methods beyond these factors are needed. Previous studies have shown that tumor histological grade is also correlated with the efficacy of neoadjuvant therapy. Single-cell sequencing analysis revealed increased expression of HIST1H4C in tumor cells from patients with high-grade histology in both datasets. Histone family genes were identified as key genes in breast cancer (BC), and previous studies have linked these genes to various cancer types. Copy number variations in HIST1H1B have been linked to cell development, growth, and proliferation in melanoma[ 24 ]. Amplification-dependent oncogene HIST1H3B has been reported to be overexpressed in liver cancer.[ 25 ] HIST1H3F has also been identified as a prognostic gene for laryngeal cancer.[ 26 ] Moreover, Down-regulation of histone H2A and H2B may reverse anthracycline resistance[ 27 ] and higher expression of HIST1H4C were associated with poorer overall survival for BC patients. Interestingly, histone modification profiles can serve as valuable biomarkers for classifying BC subtypes.[ 28 ] However, the predictor role of HIST1H4C in NACT is still unknown. Our results indicated that high expression of HIST1H4C correlated positively with the effectiveness of NACT despite having high-grade pathology. Conversely, after treatment, patients with low HIST1H4C expression showed better treatment outcomes. Therefore, HIST1H4C could serve as an effective indicator for predicting the efficacy of NACT. Furthermore, factors such as PR-, ER-, high ki67 expression, and higher histological grade can influence the efficacy of NACT. We also observed a relationship between HIST1H4C expression and some of these factors, with increased HIST1H4C expression before treatment associated with more lymph node metastases and higher expression in TNBC. Moreover, HIST1H4C expression after treatment associated with larger tumor size and higher histological grade. Limited by the sample size, we did not find a significant association between postoperative HIST1H4C expression and other clinical observations. However, high HIST1H4C expression both before and after treatment was linked to a worse prognosis, suggesting the importance of detecting HIST1H4C before and after treatment. To further validate our findings, we confirmed our results in the Curtis database due to the limited number of patients in our center. We found that high HIST1H4C expression was not only associated with increased higher level of histological grade, but also with TNBC, ER-, PR- and high Ki67 expression. Additionally, HIST1H4C expression was positively correlated with tumor volume and the number of lymph node metastases, both of those impact the efficacy of NACT. Patients with high HIST1H4C expression had a significantly poorer clinical prognosis. These results suggest that HIST1H4C expression can serve as an effective indicator for clinical outcomes. Since the 21-gene recurrence score, 70-gene MammaPrint Assay, and the PAM50 prognostic model are limited to a specific subtype or lymph node-negative breast cancer or patients at high clinical risk from breast cancer with limited predictive accuracy of the C-index, mRNA HIST1H4C expression Assay is accurate, cost-efficient, convenient, and readily available in hospitals in developing countries. However, our study also has limitations for it is a retrospective study, so there is selection bias. We did our best to include all eligible patients with NACT and obtain puncture and surgical specimens from all patients as much as possible to minimize selection bias. In conclusion, the mRNA HIST1H4C expression Assay is a highly effective tool to predict the response and prognosis of NACT, enabling the identification of patients with breast cancer who may benefit from NACT. Notably, based on analysis of HIST1H4C expression in Curtis database, we found that patients with a low level of HIST1H4C expression were more likely to gain a better OS. Consequently, mRNA HIST1H4C expression Assay may be useful for individualized NACT and follow-up. Declarations CONFLICT OF INTEREST : The authors have no relevant financial or non-financial interests to disclose. FUNDING: This work is supported by grants from the National Natural Science Foundation of China (82203122, 82201522, 82271650, 82102791, 82172917), the Foundation of Guangzhou Women and Children's Medical Center (2023BS036, 2021BS023). Guangzhou Science and Technology, Basic and Applied Basic Research Topic (Young Doctor "Sailing" Project) (2024A04J4987). Author Contribution LJ and CK conceived of the study and participated in the design of article. LQ、GR、ZHH、LYD and ZCR conducted the data analysis and drafted the article. ZHH、CYX、YX、ZXF、LJJ、LYD and ZCH collected the Tumor samples. LQ and CK performed the correction of the language and revision. All authors read and approved the final manuscript. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71(3):209–249 Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P, Ruddy K, Tsang J, Cardoso F (2019) Breast cancer. Nat Rev Dis Primers 5(1):66 Loibl S (2015) Neoadjuvant treatment of breast cancer: maximizing pathologic complete response rates to improve prognosis. Curr Opin Obstet Gynecol 27(1):85–91 Rastogi P, Anderson SJ, Bear HD, Geyer CE, Kahlenberg MS, Robidoux A, Margolese RG, Hoehn JL, Vogel VG, Dakhil SR et al (2008) Preoperative chemotherapy: updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27. 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Cancer Lett 307(1):37–46 Braunstein M, Liao L, Lyttle N, Lobo N, Taylor KJ, Krzyzanowski PM, Kalatskaya I, Yao CQ, Stein LD, Boutros PC et al (2016) Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer. Breast Cancer Res 18(1):16 Xie W, Zhang J, Zhong P, Qin S, Zhang H, Fan X, Yin Y, Liang R, Han Y, Liao Y et al (2019) Expression and potential prognostic value of histone family gene signature in breast cancer. Exp Ther Med 18(6):4893–4903 Additional Declarations No competing interests reported. Supplementary Files supfile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4525730","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315428468,"identity":"a8afcc2e-a61b-44f6-b9eb-43447cdae685","order_by":0,"name":"Li Qian","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Qian","suffix":""},{"id":315428469,"identity":"4a5fd112-58be-43e7-afcf-792c76d15695","order_by":1,"name":"Rui Ge","email":"","orcid":"","institution":"Huadong Hospital Affiliated to Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Ge","suffix":""},{"id":315428470,"identity":"1f62f600-1325-4889-bd6a-e87a5494ecd2","order_by":2,"name":"Zhu Cairong","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Cairong","suffix":""},{"id":315428471,"identity":"141bbe66-4566-43b8-aed9-0a3ce827bcd3","order_by":3,"name":"Zhong Haihu","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Haihu","suffix":""},{"id":315428472,"identity":"ab04509d-ddd4-44b1-a983-fe77397c4944","order_by":4,"name":"Cai Yuanxuan","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cai","middleName":"","lastName":"Yuanxuan","suffix":""},{"id":315428473,"identity":"8fc02eb2-1425-45ea-8eb9-5b9196e58f71","order_by":5,"name":"Liao Yongdong","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liao","middleName":"","lastName":"Yongdong","suffix":""},{"id":315428474,"identity":"36ee35a9-3266-4784-80c6-83f43345d7ec","order_by":6,"name":"Zhu Xiaofeng","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Xiaofeng","suffix":""},{"id":315428475,"identity":"c2cb8462-5e6c-4d95-9ad5-eee8c288dc64","order_by":7,"name":"Li jingjing","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"jingjing","suffix":""},{"id":315428476,"identity":"81de4aff-c8ca-4d8a-b673-c51429c10ad6","order_by":8,"name":"Ye Xuan","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Xuan","suffix":""},{"id":315428477,"identity":"441b86f2-2ea1-4dbf-a47a-cad4d380c9f1","order_by":9,"name":"Chen Kai","email":"","orcid":"","institution":"Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Kai","suffix":""},{"id":315428478,"identity":"0e6e00c9-44cc-47c6-ae3a-e694eca2d509","order_by":10,"name":"Li Jie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACPmaGhAMMFTb1/OwNRGphA2s5k5Yg2XOAWC0ggrHtcILBjQRitbAzPDzwge1wnuTM59ckfubYMfBLH79A0GEHZ/CkF/NL55RJ9m5LZpDsyykgqOUwj4Q148zZOWm3GbcdYDA4w4PfiWAtfwyYGTfcPEOKFoYE58QNN9iPQbWwHyDsl54DacaSPTnsP4F+4ZHs4cGrg4Gf/0zyh5//bOT42Y8/Nvi5zU6On4f9AX49DHCX8xiASRgDD4C7HG44QVtGwSgYBaNghAEA5e5IM3/blocAAAAASUVORK5CYII=","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Jie","suffix":""}],"badges":[],"createdAt":"2024-06-04 06:44:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4525730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4525730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58756460,"identity":"31c7c65e-0ecc-4e88-a89c-1381df669c42","added_by":"auto","created_at":"2024-06-20 17:20:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90471,"visible":true,"origin":"","legend":"\u003cp\u003eBaseline HIST1H4C expression in the good- and poor-responder groups(A); High pathological grade and low pathological grade groups(B); T1-2 and T3 groups (C); N0 and N0-3 groups (D); ER- and ER+ groups (E); PR- and PR+ groups (F); HER2- and HER2+ groups (G); Subtype of luminal, HER2+ and TNBC groups (H) and Ki67 high and low groups (I). The Kaplan–Meier curves for PFS (J) and OS (K) of patients in baseline HIST1H4C high and low groups. Data are presented as means ±SEM. *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001 by Mann-Whitney U-test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4525730/v1/4e844610e2bdeb2a14d7960f.png"},{"id":58757306,"identity":"a5e08e5e-082a-4aaa-bea0-6a9f9b65a4fc","added_by":"auto","created_at":"2024-06-20 17:28:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99344,"visible":true,"origin":"","legend":"\u003cp\u003eAfter-treatment HIST1H4C expression in the good- and poor-responder groups(A); High pathological grade and low pathological grade groups(B); T0-1 and T2-3 groups (C); N0 and N0-3 groups (D); ER- and ER+ groups (E); PR- and PR+ groups (F); HER2- and HER2+ groups (G); Subtype of luminal, HER2+ and TNBC groups (H) and Ki67 high and low groups (I). The Kaplan–Meier curves for PFS (J) and OS (K) of patients in baseline HIST1H4C high and low groups. Data are presented as means ±SEM. *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001 by Mann-Whitney U-test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4525730/v1/12be23d0ff424628e7e2541b.png"},{"id":58756462,"identity":"abeb702d-43b0-427a-9a41-d40823c37f7e","added_by":"auto","created_at":"2024-06-20 17:20:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79874,"visible":true,"origin":"","legend":"\u003cp\u003eHIST1H4C expression before and after treatment in all patients (A), in good responder (B) and in poor responder (C). HIST1H4C expression changes in High pathological grade and low pathological grade groups (D); pyT0-1 and pyT2-3 groups (E); pyN0 and pyN1-3 groups (F); Ki67 high and low groups (G). The Kaplan–Meier curves for PFS (H) and OS (I) of patients in HIST1H4C decrease and non-decrease groups. Data are presented as means ±SEM. *, P \u0026lt; 0.05; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001 by Mann-Whitney U-test.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4525730/v1/d657574d2dccdc356acc13e9.png"},{"id":60028286,"identity":"07de2e1f-bf82-4d41-ae8b-8b7050233839","added_by":"auto","created_at":"2024-07-10 18:22:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":759996,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4525730/v1/b3bba680-d47c-42ee-b31b-63e148f526a4.pdf"},{"id":58756463,"identity":"77af7049-6eeb-4227-9257-ce58f096f625","added_by":"auto","created_at":"2024-06-20 17:20:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2011249,"visible":true,"origin":"","legend":"","description":"","filename":"supfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4525730/v1/9eb5479f63cc475bef698ac6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prognostic role of HIST1H4C expression and neoadjuvant therapy efficacy in breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most common malignant tumor in women worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Chemotherapy plays a crucial role in the systemic treatment of breast cancer, with neoadjuvant chemotherapy (NAC) being particularly important. NAC aims to shrink tumor size before surgery, making inoperable breast cancer operable and improving surgical success rates. It also provides valuable information on drug sensitivity and guides subsequent treatment.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Studies have shown that NAC can improve prognosis, increase the rate of pathological complete response (pCR), and enhance event-free survival (EFS) in breast cancer patients.[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e–\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn 2014, the CTNeoBC study validated that achieving pCR following NAC was associated with a more favorable prognosis.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] The I-SPY2 study further confirmed that hormone receptor (HR) -positive breast cancer patients who achieved pCR had significantly prolonged survival.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] However, a significant number of HR-positive breast cancer patients exhibit chemotherapy resistance, and NAC may lead to an inadequate tumor shrinkage or downstaging and unexpected and irreversible adverse reactions. Therefore, identifying patients who benefit most from NAC and tailoring personalized treatment strategies is crucial.\u003c/p\u003e \u003cp\u003eTumor heterogeneity is reflected in tumor pathology grade[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], various classification systems are used globally, with the Histological Classification System (HCS) being one of the most widely recognized. Histologically, the malignancy level of breast cancer is closely associated with its grade, which correlates with the proliferation index and DNA ploidy[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As a result, this index holds considerable significance in assessing the differentiation level and prognosis of breast cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Invasive ductal carcinoma, in particular, exhibited significantly more responsiveness to chemotherapy compared to non-invasive ductal carcinoma, and treatment approaches targeting higher histological grades were notably more effective than those targeting lower grades. Studies demonstrated a positive correlation between histological grade and the pCR rate.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Therefore, histological grade serves as a crucial predictor of the response to NAC.\u003c/p\u003e \u003cp\u003eOur previous research involved analyzing single-cell sequencing data sets from GSE161529 and GSE176078. We obtained 166298 and 84320 scRNA-seq data points from 58 breast cancer samples after filtrating by nCount_RNA \u0026gt; 1000 and nCount_RNA \u0026lt; 10000 in 2 database above (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). Epithelial cells primarily expressed EPCAM, KRT7, KRT8 and patients were divided into low-grade (I-II) and high-grade (III) histological grade groups for further analysis. Tumor cells were classified into 6 subgroups, with cluster-1 showing increased proportion in the high-grade histological grade subgroup (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-F). Analysis revealed high expression of HIST1H4C in cluster-1 in both 2 dataset (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG-H), and the trajectory of differentiation in tumor cells predicted by monocle 3 showed cluster-1 may be the origin of tumor cells (Figure. S1I-J), indicating its importance in tumor development.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate the prognostic role of HIST1H4C and its interaction with the efficacy of neoadjuvant therapy and prognosis in breast cancer patients.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003emRNA extraction and Quantitative PCR with reverse transcription\u003c/p\u003e\u003cp\u003eTRIzol® Reagent (Life Technologies, USA) was used to extract total RNA from tumor tissue. RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (RR036A, Takara). Real-time quantitative PCR was performed using TB Green Premix Ex Taq II (RR820A, Takara) according to the manufacturer’s recommendations. The reactions were carried out in the LightCycler480 system with gene-specific primers. Standard dilution series were utilized to estimate the efficiency of the hic assay. Threshold cycles (Ct) were employed to calculate mRNA expression compared to the respective housekeeping gene GAPDH by relative quantification using the 2exp\u003csup\u003e−ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003ePatients in roll\u003c/p\u003e\u003cp\u003eThe study included female patients diagnosed with stage II breast cancer who underwent neoadjuvant chemotherapy (NAC) at Guangzhou Women and Children's Medical Center from August 2019 to November 2022. Inclusion criteria were: women aged 18 or older who signed an informed consent form, non-metastatic breast cancer patients with TNM stage II and above, no prior chemotherapy, endocrine therapy, surgery, or radiotherapy, normal cardiac function, ECOG score ≤ 2, and good organ function. Exclusion criteria were: patients with other malignancies in the last 5 years, or not suitable for chemotherapy. The study was a retrospective observational clinical study with the primary endpoint being pathological complete response (pCR) after NAC. Secondary endpoints included breast preservation surgery rate and prognosis-free survival (PFS) and overall survival (OS). Data collection included diagnosis confirmation, imaging examinations, molecular typing, chemotherapy, and surgical outcomes evaluation. Discrepancies of case inclusion were conferred by a principal investigator. The flowchart describing patient screening is shown in Figure S2.\u003c/p\u003e\u003cp\u003eTreatment protocol\u003c/p\u003e\u003cp\u003eThe NAC regimen was adriamycin (60 mg/m\u003csup\u003e2\u003c/sup\u003e) + cyclophosphamide (600 mg/m\u003csup\u003e2\u003c/sup\u003e) every 3 weeks for 4 cycles, followed by paclitaxel (80 mg/m\u003csup\u003e2\u003c/sup\u003e) for 12 weeks or docetaxel (75 mg/m\u003csup\u003e3\u003c/sup\u003e) and cyclophosphamide (600 mg/m\u003csup\u003e3\u003c/sup\u003e) for four to six cycles every three weeks. HER2 + BC patients were treated with double-target therapy (trastuzumab plus pertuzumab). The therapeutic efficacy of the regimens was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST). pCR was defined as the absence of any residual invasive carcinoma or ductal carcinoma in situ (DCIS) in the breast or lymph node tissue removed after NAC and examined under a microscope. Breast- or lymph node-only pCR was defined as the absence of any residual invasive cancer or DCIS confined to the breast or armpit, as determined by examination under a microscope[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Partial response (PR) was defined as a reduction of at least 30% in the sum of the longest diameter of the target lesion found by clinical examination and imaging. Progressive disease (PD) was defined as an increase of at least 20% in the sum of the longest diameter of the target lesion found by clinical examination and imaging. Stable disease (SD) was defined as neither sufficient shrinkage to qualify for PR nor sufficient growth to qualify for PD. pCR, which refers only to the breast and lymph nodes, was assessed only in patients with clinically positive lymph nodes at the time of diagnosis. Patients with pCR, breast/lymph node-specific pCR, and PR were classified as having treatment-sensitive BC, and patients with SD and PD were classified as having treatment-resistant BC.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eA completely randomized, balanced design was used for all experiments. All comparison groups have the similar variance. Wilcoxon matched-pairs signed rank test or the Mann-Whitney U-test were used to test the significance of differences in various molecular, cellular, and physiological parameters between the means or medians in treatment and control groups. A P value less than 0.05 was considered significant. Error bars in the experiments indicate standard deviation (SD) for a minimum of three independent experiments.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatients in roll\u003c/h2\u003e \u003cp\u003eIn order to investigate the correlation between HIST1H4C expression and the efficacy of neoadjuvant therapy (NACT) in breast cancer, we conducted a prospective study involving patients with newly diagnosed who had not received NACT before. A total of 109 patients were recruited from the Thyroid and Breast Department, the Guangzhou Women and Children's Medical Center in China between 2019 and 2022. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Guangzhou Women and Children's Medical Center. Detailed clinicopathological information of all patients is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The age of the enrolled patients ranged from 34 to 66 years, with 62 being premenopausal, 38 postmenopausal, and 5 whose menopausal status could not be determined due to oophorectomy. The median follow-up duration for this study, up to the cut-off date of May 1st, 2024, ranged from 16 to 63 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of HIST1H4C before treatment related to good response to neoadjuvant therapy\u003c/h2\u003e \u003cp\u003eHigh HIST1H4C expression before treatment is associated with a poor response to neoadjuvant therapy. HIST1H4C expression levels were determined using qPCR, with the median fold change of mRNA expression/GAPDH before treatment being 0.0023. Patients were classified as having a better response (pathologic complete response (pCR) or partial response (PR)) or a poor response (stable disease (SD) or progressive disease (PD)) to NACT. Results showed significantly higher HIST1H4C expression in good responders compared to poor responders (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The preoperative tumor grade, expression of ER, PR, HER2, and ki67 were determined by two physicians and we gained all tumor size messages before treatment through MR or ultrasonography. Though we only obtained pathological grade data of partly patients, 33 were evaluated as high grade (3) and 16 people were low grade (1\u0026ndash;2). Within the limited samples, we analyzed the relationship between HIST1H4C expression and pathology grade and found pathological grade revealed no significant association between HIST1H4C expression (p\u0026thinsp;=\u0026thinsp;0.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, our data also indicated baseline HIST1H4C expression differed in stage (T2 VS T3) and nodal status (N0 VS N1-3), We found higher HIST1H4C expression was positively correlated with lymph node metastasis but not with tumor size before treatment. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Furthermore, HIST1H4C expression was found to be significantly higher in triple-negative breast cancer (TNBC) samples compared to other subtypes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H) Despite previous findings linking increased Ki67 expression to chemotherapy sensitivity, our results did not show an association between HIST1H4C and Ki67 levels. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Kaplan-Meier analysis demonstrated that patients with high HIST1H4C expression before treatment had a poorer PFS to compared to those with low expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ) Finally, baseline HIST1H4C levels showed a trend towards correlation with overall survival (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of HIST1H4C after treatment related to poor response to neoadjuvant therapy\u003c/h2\u003e \u003cp\u003eTo further screen HIST1H4C expression after-treatment, qPCR also be conducted by tumor tissue HIST1H4C after-treatment. The median fold change of mRNA expression/GAPDH after-treatment was 0.0007. Firstly, the result suggests that higher expression of HIST1H4C after-treatment significantly related to poor response to NACT which is opposite to before-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Next, we analyzed the relationship between HIST1H4C expression and pathological grade after treatment and found higher pathological grade revealed significantly association to higher HIST1H4C expression. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) And tumor size and lymph node status messages after treatment was evaluated pathologically, different conclusion in some of clinical data was drawn out, as higher HIST1H4C expression after-treatment was positively correlated to larger tumor size, but the relationship between HIST1H4C after-treatment and lymph node status was unclear. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D) Moreover, in our cohort, a small subset exhibited changes in pathological subtypes after treatment. Due to the inability to evaluate the ER, PR, HER2 and Ki67 post-treatment for T0/is patients, we assumed that the pathological analysis after treatment remained consistent as pre-treatment. Analysis using the chi-square test revealed HIST1H4C expression was higher in PR-negative or HER2-negative patients. (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) Furthermore, an unpaired rank-sum test demonstrated a significant increase in HIST1H4C expression in PR-negative patients, although no such difference was observed for HER2-negative patients compared to HER2-positive patients. Neither comparison method found association between HIST1H4C expression and ER expression. Subsequently, following NACT, no significant difference in HIST1H4C expression was found among these subtypes in the postoperative specimens. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-H) Similarly, the relationship between post-treatment HIST1H4C expression and Ki67 level remains inconclusive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Importantly, our findings suggest that high post-treatment HIST1H4C expression is associated with a poorer PFS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). And finally, high post-treatment HIST1H4C showed a trend towards correlation with overall survival (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11). (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe data above indicates that both baseline and post-NACT HIST1H4C could be important indicators in predicting the efficacy of NACT. HIST1H4C may be regulated by NACT, and its expression could potentially predict clinical prognosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDecimated HIST1H4C correlated with good response to neoadjuvant therapy\u003c/h2\u003e \u003cp\u003eTo investigate whether HIST1H4C may be regulated by NACT, a comparison between baseline and post-treatment was conducted. Paired t-tests revealed a significant downregulation of HIST1H4C by NACT. Patients who responded well showed a notable decrease in HIST1H4C expression, while patients with a poor response did not exhibit a significant decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). An effective decrease in HIST1H4C was defined as a reduction of over 50% as Decrease group, while a reduction of less than 50% or an increase was categorized as the Non-decrease group. Subsequent analysis showed that patients with lower grade levels (2), earlier stages (T2), less lymphatic metastasis (N0), or higher Ki67 expression did not demonstrate a more pronounced decimation of HIST1H4C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-G). Unfortunately, the data did not show a significant difference in Kaplan-Meier analysis between the decrease group and non-decrease group, indicating that patients with a significant reduction in HIST1H4C did not have improved progression-free survival (PFS) or overall survival (OS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of HIST1H4C related to poor prognosis in CURTIS\u003c/h2\u003e \u003cp\u003eTo determine the relationship between HIST1H4C expression and clinical data such as pathological grade and clinical prognosis, medical records were obtained from the CURTIS database. Analysis revealed a significant increase in HIST1H4C expression in high-grade (3) compared to low-grade (1\u0026ndash;2) subgroups. (Fig.S3A) Additionally, the expression of HIST1H4C was found to be significantly associated with tumor size and lymph node metastasis in patients with higher levels of expression. Subpopulations in the CURTIS database was further divided based on CLAUDIN_SUBTYPE, including luminal A, luminal B, HER2+, TNBC. Comparison of HIST1H4C expression in triple-negative breast cancer (basal, normal, or claudin-low) indicated increased expression compared to luminal and HER2\u0026thinsp;+\u0026thinsp;subsets, with no significant difference observed in the latter two subgroups. Moreover, higher expression of HIST1H4C was observed in ER-negative, PR-negative patients, with no significant difference in HIST1H4C expression seen in the HER2-negative and positive subgroups. (Fig.S3B and Table S2) Subsequent analysis by dividing patients into two groups based on median HIST1H4C levels revealed significantly shorter survival times in patients with high HIST1H4C expression, highlighting the potential prognostic value of HIST1H4C expression. (Fig.S3C)\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBreast cancer is the most common malignancy globally and a leading cause of cancer-related deaths in women[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Some breast cancers present with large tumor and metastatic lymph nodes at diagnosis, leading to a low 5-year survival rate. In the pursuit of optimal care, clinicians are striving to identify areas where 'downgrade' treatment can be effective[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. pCR has long been associated with a better prognosis in patients undergoing NAC[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although numbers potential benefits of NACT, the risks of delaying surgery with poor treatment response could develop unresectable disease and increased chances for metastatic tumor spread. In addition, the toxic effects of NACT might aggravate morbidity or further delay surgical resection, and increasing surgical risk.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Thereby, it is greatly need to develop a specific biomarker signature to define the ideal patient population for NAC.\u003c/p\u003e \u003cp\u003eVarious factors such as molecular typing, Ki67 expression, histological grade, tumor size, lymph node metastasis, and immune cell infiltration have been linked to the efficacy of NACT in breast cancer.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] However, in the era of personalized therapy, additional methods beyond these factors are needed.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that tumor histological grade is also correlated with the efficacy of neoadjuvant therapy. Single-cell sequencing analysis revealed increased expression of HIST1H4C in tumor cells from patients with high-grade histology in both datasets. Histone family genes were identified as key genes in breast cancer (BC), and previous studies have linked these genes to various cancer types. Copy number variations in HIST1H1B have been linked to cell development, growth, and proliferation in melanoma[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Amplification-dependent oncogene HIST1H3B has been reported to be overexpressed in liver cancer.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] HIST1H3F has also been identified as a prognostic gene for laryngeal cancer.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Moreover, Down-regulation of histone H2A and H2B may reverse anthracycline resistance[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and higher expression of HIST1H4C were associated with poorer overall survival for BC patients. Interestingly, histone modification profiles can serve as valuable biomarkers for classifying BC subtypes.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] However, the predictor role of HIST1H4C in NACT is still unknown.\u003c/p\u003e \u003cp\u003eOur results indicated that high expression of HIST1H4C correlated positively with the effectiveness of NACT despite having high-grade pathology. Conversely, after treatment, patients with low HIST1H4C expression showed better treatment outcomes. Therefore, HIST1H4C could serve as an effective indicator for predicting the efficacy of NACT. Furthermore, factors such as PR-, ER-, high ki67 expression, and higher histological grade can influence the efficacy of NACT. We also observed a relationship between HIST1H4C expression and some of these factors, with increased HIST1H4C expression before treatment associated with more lymph node metastases and higher expression in TNBC. Moreover, HIST1H4C expression after treatment associated with larger tumor size and higher histological grade. Limited by the sample size, we did not find a significant association between postoperative HIST1H4C expression and other clinical observations. However, high HIST1H4C expression both before and after treatment was linked to a worse prognosis, suggesting the importance of detecting HIST1H4C before and after treatment.\u003c/p\u003e \u003cp\u003eTo further validate our findings, we confirmed our results in the Curtis database due to the limited number of patients in our center. We found that high HIST1H4C expression was not only associated with increased higher level of histological grade, but also with TNBC, ER-, PR- and high Ki67 expression. Additionally, HIST1H4C expression was positively correlated with tumor volume and the number of lymph node metastases, both of those impact the efficacy of NACT. Patients with high HIST1H4C expression had a significantly poorer clinical prognosis. These results suggest that HIST1H4C expression can serve as an effective indicator for clinical outcomes. Since the 21-gene recurrence score, 70-gene MammaPrint Assay, and the PAM50 prognostic model are limited to a specific subtype or lymph node-negative breast cancer or patients at high clinical risk from breast cancer with limited predictive accuracy of the C-index, mRNA HIST1H4C expression Assay is accurate, cost-efficient, convenient, and readily available in hospitals in developing countries. However, our study also has limitations for it is a retrospective study, so there is selection bias. We did our best to include all eligible patients with NACT and obtain puncture and surgical specimens from all patients as much as possible to minimize selection bias.\u003c/p\u003e \u003cp\u003eIn conclusion, the mRNA HIST1H4C expression Assay is a highly effective tool to predict the response and prognosis of NACT, enabling the identification of patients with breast cancer who may benefit from NACT. Notably, based on analysis of HIST1H4C expression in Curtis database, we found that patients with a low level of HIST1H4C expression were more likely to gain a better OS. Consequently, mRNA HIST1H4C expression Assay may be useful for individualized NACT and follow-up.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e CONFLICT OF INTEREST\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFUNDING:\u003c/h2\u003e \u003cp\u003eThis work is supported by grants from the National Natural Science Foundation of China (82203122, 82201522, 82271650, 82102791, 82172917), the Foundation of Guangzhou Women and Children's Medical Center (2023BS036, 2021BS023). Guangzhou Science and Technology, Basic and Applied Basic Research Topic (Young Doctor \"Sailing\" Project) (2024A04J4987).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLJ and CK conceived of the study and participated in the design of article. LQ、GR、ZHH、LYD and ZCR conducted the data analysis and drafted the article. ZHH、CYX、YX、ZXF、LJJ、LYD and ZCH collected the Tumor samples. LQ and CK performed the correction of the language and revision. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71(3):209\u0026ndash;249\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P, Ruddy K, Tsang J, Cardoso F (2019) Breast cancer. Nat Rev Dis Primers 5(1):66\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoibl S (2015) Neoadjuvant treatment of breast cancer: maximizing pathologic complete response rates to improve prognosis. 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Exp Ther Med 18(6):4893\u0026ndash;4903\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HIST1H4C, neoadjuvant therapy efficacy, prognosis, BC","lastPublishedDoi":"10.21203/rs.3.rs-4525730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4525730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ebreast cancer is a common and highly malignant, currently, HIST1H4C was found to be associated with several human malignancies. The purpose of this study is to investigate tissue HIST1H4C expression in breast cancer and explore its role in disease progression and its interaction with neoadjuvant therapy efficacy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ewe analyzed tissue HIST1H4C mRNA expression in BC tissue samples from 105 patients received with neoadjuvant therapy using qPCR between 2019\u0026ndash;2022.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eStatistical analysis showed that a high expression of HIST1H4C before neoadjuvant therapy was positively related to good responder (CR\u0026thinsp;+\u0026thinsp;PR), while high expression of HIST1H4C after neoadjuvant therapy was negatively related good responder. And HIST1H4C expression was significantly decreased in patients with good responder. In addition, high HIST1H4C expression was also related to ER negative, PR negative, high KI67 expression, high level of histological grade, large tumor size and more lymph node metastases in Curtis database. Furthermore, high HIST1H4C expression before and after-treatment in our center or in database has a positively correlation with poor prognosis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHIST1H4C is the potential biomarker of neoadjuvant therapy and prognosis for breast cancer.\u003c/p\u003e","manuscriptTitle":"Prognostic role of HIST1H4C expression and neoadjuvant therapy efficacy in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 17:20:28","doi":"10.21203/rs.3.rs-4525730/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adfbdb62-dd6a-4baa-b2cd-00391c8c68aa","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-24T06:44:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 17:20:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4525730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4525730","identity":"rs-4525730","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}