Neo-Family History Score Is A Novel Biomarker of Pathological Complete Response, Safety, and Survival Outcomes In Patients With Breast Cancer Receiving Neoadjuvant Platinum-Based Chemotherapy: A Retrospective Analysis of Two Prospective Trials

Background: Homologous recombination repair gene mutations are associated with increased platinum-based chemosensitivity, whereas few studies have reported the predictive value of family history of cancer for breast cancer in the neoadjuvant setting. This study aimed to construct a brief and effective novel family history scoring system and explore its association with pathological complete response (pCR), survival outcomes, and safety for locally advanced breast cancer receiving platinum-based neoadjuvant chemotherapy. Methods: A total of 262 patients treated with neoadjuvant cisplatin and paclitaxel were included. Neo-Family History Score (NeoFHS) was calculated according to cancer type, age at diagnosis, kinship, and number of affected relatives. Logistic regression was performed to analyze the association between pCR and NeoFHS. Survival rates were compared by Kaplan-Meier curves, examined by log-rank test and Cox proportional hazard regressions. Results: For all patients enrolled in this study, clinical tumor stage (p=0.048), estrogen receptor status (p=0.001), progesterone receptor status (p=0.036), human epidermal growth factor receptor 2 (HER2) status (p=0.013), and molecular subtype (p=0.016) were signicantly related to NeoFHS. The multivariate logistic regression revealed that NeoFHS is an independent predictive factor of pCR (OR=2.262, 95% CI 1.159-4.414, p=0.017), especially in node-positive (OR=3.088, 95% CI 1.498-6.367, p=0.002), hormone receptor-positive (OR=2.645, 95% CI 1.164-6.010, p=0.020), and HER2-negative subgroups (OR=4.786, 95% CI 1.550-14.775, p=0.006). Kaplan-Meier estimates suggested that NeoFHS could serve as an independent prognostic factor for relapse-free survival in the whole group (adjusted HR=0.305, 95% CI 0.102-0.910, p=0.033) pCR. The least absolute shrinkage and selection operator (LASSO) and 10-fold cross validation were used to select optimal predictive parameters. Receiver operating characteristic (ROC) curves, decision curve analysis (DCA), and clinical impact curves (CICs) were performed to investigate if NeoFHS could promote the ability to predict individual response to NAC. Nomograms were established to display the predicted probabilities of pCR. Calibration curve was examined by Hosmor-Lemeshow test. The estimated median follow-up was calculated using the reverse Kaplan-Meier method. Survival rates were compared by Kaplan-Meier curves, examined by log-rank test. Cox proportional hazard regressions were performed to derive hazard ratios (HRs) with 95% CIs. The adjustment factors were baseline clinicopathological variables including menopausal status, clinical T stage, clinical nodal status, HorR status, HER2 status, Ki67 index, and BMI. All analyses were performed R The results were considered signicant with p<0.05.

Conclusions: Our study revealed that NeoFHS is a practical and effective biomarker for predicting not only pCR and survival outcomes but also chemotherapy-induced AEs for neoadjuvant platinum-based chemotherapy for breast cancer. It may help screen candidate responders and guide safety managements in the future.

Background
Over the last decades, neoadjuvant chemotherapy (NAC) has become a standard management for patients with locally advanced breast cancer. It has been revealed that patients with pathological complete response (pCR) have improved clinical outcomes including disease-free survival (DFS) and overall survival (OS), [1] while failure to achieve pCR is the strongest independent risk factor for recurrence. [2] Platinum is a classical cytotoxic agent that results in DNA double-strand breaks (DSBs) and subsequently programs cell death under failure to repair or excessive damage accumulation. [3,4] Cisplatin-based chemotherapy could induce superior response in locally advanced breast cancer. [5][6][7][8] Our prior research showed that patients with locally advanced breast cancer achieved an encouraging pCR rate (34.4%) after receiving neoadjuvant cisplatin plus paclitaxel. Much more exciting data was observed in those with human epidermal growth factor receptor 2 (HER2)-positive breast cancer (52.4%) and triple-negative breast cancer (TNBC; 64.7%). Even so, a considerable number of patients still encounter the failure to achieve pCR. [9] To well distinguish those who respond from those who do not in the neoadjuvant setting, research is warranted to investigate the potential biomarkers for individual chemosensitivity at the initial diagnosis.
Platinum resistance will occur once DSBs triggers excessive DNA damage repair, [10] of which homologous recombination repair (HRR) is the major process. [11] Previous evidence has revealed that platinum-based chemosensitivity is associated with mutation of genes involved in HRR, especially BRCA1/2. [12,13] The GeparSixto trial revealed that homologous recombination de ciency (HRD) independently predicts pCR in TNBC, and improved pCR rate was observed for homologous recombination de cient tumors by adding carboplatin to paclitaxel and nonpegylated liposomal doxorubicin (PMCb). [14] Of note, HRD is not capable of explaining all the familial breast cancer. Generally, HRR gene mutations are identi ed in only about 20% of breast cancer patients with family history of breast cancer. [15,16] On the other hand, family history may re ect changes of hereditary substances beyond HRR gene mutations, including variants in other protein-coding genes [15] as well as non-coding RNAs, [17] epigenetic regulation, [18] and some unrecognized mechanisms. The previous study by David et al. suggested that family history of breast, ovarian, or pancreatic cancer might serve as a predictive marker for rst-line platinum treatment in metastatic pancreatic adenocarcinoma. [19] Regrettably, it remains unclear whether family history could predict chemosensitivity for breast cancer patients especially those treated with platinum-based regimen.
So far, traditional methods of evaluating family history usually aim to assess incidence, [20,21] mortality, [22] and BRCA mutation risk [23] instead of chemosensitivity for patients with breast cancer. And those de nitions merely include BRCA-related cancers in the kindreds. Notably, at most 55% of breast cancer with family history of both breast cancer and ovarian cancer can be explained by the high-penetrance BRCA1/2 variants. [24] It hints that many other breast cancer susceptibility genes also contribute. [15] Interestingly, patients with family history of cancer other than breast or ovarian cancer generally have HER2-positive disease. [25] Therefore, we hypothesized that family history of BRCA-related cancers and non-BRCA cancers might both in uence the biological features of breast cancer. Here we proposed a brief quantitative novel scoring system named Neo-Family History Score (NeoFHS) and postulated that it might serve as a predictive biomarker of platinum-based chemosensitivity for breast cancer in the neoadjuvant setting. In this study, we retrospectively investigated the predictive and prognostic value of NeoFHS in patients from our platinum-based prospective neoadjuvant trials.

Patients and study design
We performed a retrospective study on women with T 2-4 N 0-3 M 0 breast cancer enrolled in two prospective neoadjuvant clinical trials, separately registered as SHPD001 (ClinicalTrials.gov identi er: NCT02199418) and SHPD002 (ClinicalTrials.gov identi er: NCT02221999). All the patients were from independent families. Ethical approvals were granted for both trials by the Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University. All the enrolled patients signed written informed consents covering translational research.
Details of the protocols were published previously. [9] In brief, paclitaxel 80 mg/m² was intravenously given on day 1, 8, 15, and 22, combined with cisplatin 25 mg/m² on day 1, 8, and 15 every 28 days for 4 cycles. For HER2-positive patients, trastuzumab was recommended concurrently at a loading dose of 4 mg/kg followed by a maintenance dose of 2 mg/kg, weekly, for 16 weeks. Hormone receptor (HorR)positive patients in SHPD002 were allocated to chemotherapy with or without endocrine therapy [aromatase inhibitor for postmenopausal patients or gonadotropin-releasing hormone agonist (GnRHa) for premenopausal counterparts]. Premenopausal patients with TNBC were randomized to chemotherapy with or without GnRHa in SHPD002. Surgery was given sequentially after NAC.
Between January 2014 and January 2019, 262 patients from these two trials who had undergone breast surgery at Renji Hospital were available for the analysis. This study was presented according to the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines. [26] Data collection The baseline data was collected prospectively at enrollment. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters, and 25 was used to separate groups. The follow-up information was also prospectively collected.
All the biopsy tissues were diagnosed as invasive breast cancer by Department of Pathology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. HorR positivity was de ned as ⩾1% tumor cell nuclei were stained for estrogen receptor (ER) or progesterone receptor (PR) by immunohistochemistry (IHC). HER2 positivity was de ned as IHC 3+ or ampli cation by uorescence in situ hybridization according to the American Society of Clinical Oncology/College of American Pathologists recommendations 2013. [27] In terms of Ki67 index, we used 50% to separate groups.

Calculation of traditional family history scores
Multiple classical tools for identifying BRCA1/2 mutations were recommended by US Preventive Services Task Force (USPSTF). [23] The Ontario Family History Assessment Tool uses family data on breast, ovarian, prostate, and colon cancer according to onset age for summation, with a score of ⩾10 implicating doubling of lifetime risk for developing breast cancer (22%). The Manchester Scoring System incorporates family history of female and male breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer according to onset age, with a combined score of 15 corresponding to 10% chance to carry BRCA1/2 mutations. The Pedigree Assessment Tool includes female breast cancer with onset age, male breast cancer, ovarian cancer, and Ashkenazi Jewish heritage, with a score of ⩾8 as the optimal threshold for referral to genetic counseling. Here, we calculated these traditional family history scores for all the included patients and de ned high risk as the score ⩾1 for each tool.
Conduction of Neo-Family History Score (NeoFHS) Considering that cancer type, age at diagnosis, kinship, and affected number may have comprehensive in uence on the family background of a patient, [23,29,30] we conducted the NeoFHS system to quantify individual family history. NeoFHS = ∑ the age-, kinship-, and cancer-speci c score of the affected relative It was calculated as the total age-, kinship-, and cancer-speci c scores for all affected relatives ( Table 1).
The cut-off value of NeoFHS was determined to be 0.5, the higher quartile of all data.

Outcomes
The outcomes in this study were pCR, relapse-free survival (RFS), distant relapse-free survival (DRFS), visceral metastasis-free survival (VMFS), and safety. The de nition of pCR was no residual invasive cancer in the breast and the absence of cancer cells in lymph node samples taken at the time of surgery (ypT 0/is ypN 0 ). RFS was calculated as the time from surgery to rst occurrence of locoregional, ipsilateral, contralateral, distant recurrence, and death from any cause. DRFS was de ned as the time from surgery to rst occurrence of distant recurrence and death from any cause. VMFS referred to the time from surgery until rst occurrence of visceral metastasis and death from any cause. Adverse events (AEs) were assessed during study period and graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.01.

Bioinformatics analyses
We derived a gene expression pro le GSE75678 from the Gene Expression Omnibus (GEO), and identi ed the differentially expressed genes (DEGs) between breast cancer patients without family history of any cancer and those with family history of any cancer, or BRCA-related cancer, or non-BRCA cancer, respectively.

Baseline clinicopathological characteristics
Detailed data on family history was available from 262 patients. Among them, 69 (26.3%) patients presented with rst-degree family history of any cancer, 37 (14.1%) patients with affected second-degree relatives, and 5 (1.9%) patients with affected third-degree relatives (Fig. 1). In total, 25 patients (9.5%) were at high Ontario risk, 17 patients (6.5%) had high Manchester risk, and 21 patients (8.0%) showed high Pedigree risk. We found an inverse correlation between age and Ontario risk score (p = 0.037) as well as Pedigree risk score (p = 0.011), and a positive correlation between BMI and Manchester risk score (p = 0.009). However, none of these classical family history scores were related to the pathological features of tumors (Supplementary Table 1).
The nomogram was created for the predictive model that combined NeoFHS with the extracted clinicopathological variables (Fig. 3A). The corresponding calibration curve showed great agreement between the predicted probabilities and observed pCR outcomes (χ 2 = 10.39, p = 0.239; Fig. 3B (Fig. 3C). The DCA consistently depicted more bene ts with the model combining NeoFHS with clinicopathological features (Fig. 3D). The nomogram for predicting pCR demonstrated that cost/bene t ratios were lower with the risk threshold less than 0.7 (Fig. 3E).  Fig. 4).

Subgroup Analysis Of Pcr Rates
In the multivariate analyses, NeoFHS could serve as an independent predictive factor for pCR in T2  Table 9).
There was no interaction detected between clinicopathological variables and NeoFHS for pCR (Fig. 4).

Relapse-free Survival
The

Safety
Interestingly, Song et al. found that patients with rst-degree family history of cancer other than breast or ovarian cancer tended to have HER2-positive disease (p = 0.03). [25] Therefore, we postulated that family history of BRCA-related cancer and non-BRCA cancer might contribute to different biological characteristics. To verify this opinion, we further performed pathway analyses of the DEGs between breast cancer patients without family history of any cancer and those with family history of any cancer, or BRCA-related cancer, or non-BRCA cancer, respectively. As a result, totally different pathways were enriched under the various de nitions of family history, suggesting that family history of merely BRCArelated cancer is insu cient to re ect individual genetic background thoroughly. In the current study, we proposed a brief but comprehensive scoring system, NeoFHS, which included both BRCA-related cancer and non-BRCA cancer, as well as age at diagnosis, kinship, and affected number. Partially consistent with previous studies above and fully validating our hypothesis, our data showed that ER negativity, PR negativity, HER2 positivity, higher T stage, and molecular subtype were related to NeoFHS. All these ndings highlight the necessity and advantage to take various cancer types into comprehensive consideration for the de nitions of family history, which we did exactly in the NeoFHS system.
The current study revealed that NeoFHS could serve as an independent predictive factor for pCR in breast cancer receiving platinum-based NAC, especially in node-positive, HorR-positive, and HER2-negative patients. To the best of our knowledge, our study for the rst time substantiated that family history of cancer contributed to a better pCR rate, which signi cantly increased to 44.9% for NeoFHS-high patients from 27.7% for those with lower NeoFHS. Good performance was shown in the predictive model that combined NeoFHS with baseline clinicopathological variables. So far, emerging evidence has indicated that breast cancer arising in BRCA1/2 germline mutation carriers achieves higher response to DNAdamaging agents. Bryski et al. reported that the highest pCR rate for regimens in BRCA1-mutation carriers turned out to be 83% for cisplatin, compared with 22% for AC (doxorubicin and cyclophosphamide) or FAC ( uorouracil, doxorubicin, and cyclophosphamide), 8% for AT (doxorubicin and docetaxel), and 7% for CMF (cyclophosphamide, methotrexate, and uorouracil). [12] Concordantly, the GeparOcto trial randomized patients with breast cancer to sequential intense dose-dense epirubicin, paclitaxel, and cyclophosphamide or weekly PM, and its secondary analysis showed a higher pCR rate in patients with germline BRCA1/2 variants than those without (60.4% vs 46.7%; OR = 1.74, 95% CI 1.13-2.68, p = 0.01).
[37] Additionally, the triple-negative trial (TNT), which recruited advanced TNBC, showed a better response to carboplatin than to docetaxel in germline BRCA1/2 mutation group (objective response rate 68.0% vs 33.3%, p = 0.03), with a signi cant interaction between platinum-based regimen and BRCA1/2 mutation (p = 0.01). [38] On these premises, the classical family history scoring systems for assessing BRCA1/2 mutations have potential in pCR prediction. However, we found that Ontario, Manchester and Pedigree risk score unexceptionally failed to predict pCR in our cohorts. This is consistent with the limited evidence on the association between pCR and family history of BRCA-related cancer. Ding et al. enrolled patients with HER2-positive and node-positive breast cancer to receive neoadjuvant paclitaxel, carboplatin, and trastuzumab, and the pCR rates were 50.0% and 32.9% respectively in patients with and without rst-or second-degree family history of breast cancer (HR = 0.441, 95% CI 0.173-1.123, p = 0.086). [39] The secondary analysis of the GeparSixto trial reported that the pCR rate was 49.1% in the non-carboplatin arm and 61.4% in the carboplatin arm in TNBC with family history of breast or ovarian cancer (OR 1.65, 95% CI 0.77-3.53, p = 0.19), whereas the corresponding rate increased from 37% without carboplatin to 53.9% with carboplatin in patients without the same family history (OR = 2.0, 95% CI 1.10-3.62, p = 0.02).
[40] These facts might be due to their de nitions of family history, which did not take into account cancers except breast and ovarian cancer. It prompted us once again to focus on family history of cancer including but not limited to BRCA-related cancer. In the pathway analyses of the identi ed DEGs between patients with family history of non-BRCA cancer and those without family history of any cancer, it highlighted multiple downregulated genes including IL-10 for participating in the response to drug (Supporting information). Yang et al. found that tumor-associated macrophages could induce paclitaxel resistance via activating IL-10/STAT3/Bcl-2 signaling pathway in breast cancer. [41] Therefore, NeoFHS might well distinguish patients with hypersensitivity to cytotoxic agents from those without through the function of many other key genes beyond BRCA1/2. Further basic research is required to elucidate the essential difference between family history of BRCA-related cancer and non-BRCA cancer.
In parallel, our data substantiated the prognostic value of NeoFHS in patients treated with neoadjuvant platinum, especially in node-positive patients. more emphasis on family history of not only BRCA-related cancer but also non-BRCA cancer in chemotherapy-induced AEs. The NeoFHS system may reasonably help us with early prediction and better management for chemotherapy-induced AEs.
Our study has several limitations. First, our data was analyzed retrospectively. However, all data was collected prospectively in the registered clinical trials. Therefore, it may indicate potential intrinsic rules to some extent. Second, it was not mature to perform analysis for OS. Prolonged follow-up will be warranted. In addition, the sample size was relatively small. However, we did substantiate a profound predictive and prognostic value of NeoFHS for breast cancer treated with NAC. We will expand the sample size for further validation.

Conclusions
In summary, family history of cancer may function as a double-edged sword in both protecting cancer cells from chemoresistance and inducing side effects to normal cells. The NeoFHS system could be identi ed as a practical and effective biomarker for predicting not only chemosensitivity but also

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 that they have no competing interests.

Funding
The authors disclosed receipt of the following nancial support for the research, authorship, and/or publication of this article:     Subgroup analysis for pCR by NeoFHS Notes: ORs and 95% CIs were derived from univariate logistic regression model. Interaction p values were shown between subgroups and NeoFHS. Abbreviations: OR, odds ratio; CI, con dence interval; NeoFHS, Neo-Family History Score; T, tumor; HorR, hormone receptor; HER2, human epidermal growth factor receptor 2; BMI, body mass index. Kaplan-Meier estimates of relapse-free survival according to different family history scoring systems in all patients (A-D) and node-positive patients (E) Abbreviations: RFS, relapse-free survival; HR, hazard ratio; CI, con dence interval; NeoFHS, Neo-Family History Score.