Evaluating the prognostic significance of HIST1H4C in breast cancer: implications for neoadjuvant therapy.

Introduction
Breast cancer (BC) is the most common malignant tumor in women worldwide1. Chemotherapy plays a crucial role in the systemic treatment of breast cancer, with neoadjuvant chemotherapy (NACT) being particularly important. NACT 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 treatment2. Studies have shown that NACT can improve prognosis, increase the rate of pathological complete response (pCR), and enhance event-free survival (EFS) in breast cancer patients3–5.
In 2014, the CTNeoBC study validated that achieving pCR following NACT was associated with a more favorable prognosis6. The I-SPY2 study further confirmed that hormone receptor (HR) -positive breast cancer patients who achieved pCR had significantly prolonged survival7. However, a significant number of HR-positive breast cancer patients exhibit chemotherapy resistance, and NACT may lead to an inadequate tumor shrinkage or downstaging and unexpected and irreversible adverse reactions. Therefore, identifying patients who benefit most from NACT and tailoring personalized treatment strategies is crucial.
Tumor heterogeneity is reflected in tumor pathology grade8, 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 ploidy9. As a result, this index holds considerable significance in assessing the differentiation level and prognosis of breast cancer9,10. 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 rate11,12. Therefore, histological grade serves as a crucial predictor of the response to NACT.
Our previous research involved analyzing single-cell sequencing data sets from GSE161529 and GSE176078. We obtained 166,298 and 84,320 scRNA-seq data points from 58 breast cancer samples after filtrating by nCount_RNA > 1000 and nCount_RNA < 10,000 in 2 database above (Fig.S1A-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.S1C-F). Analysis revealed high expression of HIST1H4C in cluster-1 in both 2 dataset (Fig.S1G-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 HIST1H4C 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 NACT 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 NACT. 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 NACT regimen was adriamycin (60 mg/m2) + cyclophosphamide (600 mg/m2) every 3 weeks for 4 cycles, followed by paclitaxel (80 mg/m2) for 12 weeks or docetaxel (75 mg/m3) and cyclophosphamide (600 mg/m3) 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 NACT 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 microscope13. 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 enrolled
In order to investigate the correlation between HIST1H4C expression and the efficacy of 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. 1A). 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 (III) and 16 people were low grade (I-II). 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. 1B). 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. 1C-D). Furthermore, HIST1H4C expression was found to be significantly higher in triple-negative breast cancer (TNBC) samples compared to other subtypes (Fig. 1E-H). Despite previous findings linking increased Ki67 expression to chemotherapy sensitivity14, our results did not show an association between HIST1H4C and Ki67 levels (Fig. 1I). 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. 1J). Finally, baseline HIST1H4C levels showed a trend towards correlation with overall survival (p = 0.06) (Fig. 1K).

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. 2A). 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. 2B). 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. 2C-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. 2E-H). Similarly, the relationship between post-treatment HIST1H4C expression and Ki67 level remains inconclusive (Fig. 2I). 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. 2J-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. 3A-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. 3D-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. 3H-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 (III) compared to low-grade (I-II) 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 women15. 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 effective16,17. pCR has long been associated with a better prognosis in patients undergoing NACT6,18. 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 risk19,20. Thereby, it is greatly need to develop a specific biomarker signature to define the ideal patient population for NACT.
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 cancer12,21–24. 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.
The fundamental role of the histone H4 family in chromatin structure is crucial. Histone H4 is an integral component of the histone octamer around which DNA wraps, contributing to chromatin condensation and stability. Post-translational modifications (PTMs) of histone H4, such as acetylation, methylation, and ubiquitination, regulate key processes including gene transcription, DNA replication, and repair25. Previous studies have indicated that HIST1H1A and E2F1 are highly expressed in chemotherapy-sensitive cells26. Additionally, histone H1 kinase may promote cell cycle progression and proliferation by regulating the levels of cell cycle-related proteins27,28, while actively proliferating cells increase chemotherapy sensitivity14. Conversely, elevated levels of HIST1H1A have also been associated with platinum-based chemotherapy resistance29, and downregulation of histones H2A and H2B may reverse anthracycline resistance30. Thus, the relationship between HIST1H4C and chemotherapy efficacy is complex and contentious.
Research indicates that Titanocenes, a class of metal-based anticancer agents, inhibit cell proliferation and promote apoptosis. Notably, in tumor cells treated with Titanocenes, proteins for DNA damage repair, such as RAD50 and NEIL2, are downregulated along with histones HIST1H1D (H1.2), HIST1H4B (H4), HIST2H2AA3 (H2A1), and HIST1H2BA (H2B1A)31. This observation aligns with our findings, as our research demonstrates that HIST1H4C expression can be modulated by chemotherapy. Overall, we found that HIST1H4C expression significantly decreases following NACT. However, due to tumor heterogeneity, patients with favorable NACT responses show marked decreases in HIST1H4C expression, while those with poor responses exhibit no reductions.
Further exploration of the relationship between HIST1H4C expression and clinical prognosis. Copy number variations in HIST1H1B have been linked to cell development, growth, and proliferation in melanoma32. Amplification-dependent oncogene HIST1H3B has been reported to be overexpressed in liver cancer33. HIST1H3F has also been identified as a prognostic gene for laryngeal cancer34. Moreover, higher expression of HIST1H4C were associated with poorer overall survival for BC patients35. This is consistent with our conclusions, as higher levels of HIST1H4C expression are associated with poorer prognoses in breast cancer patients.
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.

Supplementary Information
Below is the link to the electronic supplementary material.