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Prognostic Significance of the Tumor-Stromal Ratio in Invasive Breast Cancer and a Proposal of a New Ts-TNM Staging System

Prognostic Significance of the Tumor-Stromal Ratio in Invasive Breast Cancer and a Proposal of a... Hindawi Journal of Oncology Volume 2020, Article ID 9050631, 10 pages https://doi.org/10.1155/2020/9050631 Research Article Prognostic Significance of the Tumor-Stromal Ratio in Invasive Breast Cancer and a Proposal of a New Ts-TNM Staging System 1,2 3 1,4 1,4 Qian Xu , Jing-Ping Yuan, Yuan-Yuan Chen, Hong-Yan Zhang, 1,4 1,2 Lin-Wei Wang , and Bin Xiong Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Zhongnan Hospital of Wuhan University, 4300711 Wuhan, China Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China Department of Pathology, Renmin Hospital of Wuhan University, 430060 Wuhan, China Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan, China Correspondence should be addressed to Lin-Wei Wang; wanglinweiwhu@163.com and Bin Xiong; binxiong1961@whu.edu.cn Received 7 October 2019; Revised 16 January 2020; Accepted 5 February 2020; Published 21 April 2020 Academic Editor: Vincenzo Coppola Copyright © 2020 Qian Xu et al. 2is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Previous studies have demonstrated that the tumor-stromal ratio (TSR) was an independent prognostic factor in several types of carcinomas. 2is study aimed at exploring the prognostic significance of the TSR in invasive breast cancer using immunohistochemistry (IHC)-stained tissue microarrays (TMAs) and integrating the TSR into the traditional tumor-node- metastasis (TNM) staging system. Methods. 2e prepared 7 TMAs containing 240 patients with 480 invasive BC specimens were stained with cytokeratin (CK) by the IHC staining method. 2e ratio of tumor cells and stromal cells was visually assessed. TSR > 1 and TSR≤ 1 were categorized as the high TSR (low stroma) and low TSR (high stroma) groups, respectively, and the prognostic value of the TSR at 5-year disease-free survival (5-DFS) was analyzed. A new Ts-TNM (tumor stroma-tumor-node-metastasis) staging system was established and assessed. Results. IHC staining of CK could specifically label tumor cells with clear contrast, making it easy to manually assess TSR. High TSR (low stroma) and low TSR (high stroma) were observed in 52.5% (n � 126) and 47.5 (n � 114) of the cases, according to the division of value 1. A Kaplan–Meier analysis showed that patients in the low TSR group had a worse 5-DFS compared with patients in the high TSR group (P � 0.022). Multivariable analysis indicated that the T stage (P � 0.014), N status (P< 0.001), histological grade (P< 0.001), estrogen receptor status (P � 0.015), and TSR (P � 0.011) were independent prognostic factors of invasive BC patients. 2e new Ts-TNM staging system combining TSR, tumor staging, lymph node status, and metastasis staging was established. 2e receiver operating characteristic (ROC) curve analysis dem- onstrated that the ability of the Ts-TNM staging system to predict recurrence was not lower than that of the TNM staging system. Conclusions. 2is study confirms that the TSR is a prognostic indicator for invasive breast cancer. 2e Ts-TNM staging system containing stromal and tumor information may optimize risk stratification for invasive breast cancer. metastasis still occurs in a proportion of patients [3, 4]. More 1. Introduction prognostic factors are needed to optimize invasive breast Breast cancer (BC) is the leading cause of cancer deaths cancer risk stratification to guide precise treatment. among females in both developed and developing countries. Currently, the tumor-node-metastasis (TNM) staging With an estimated 1.6 million cases and 520,000 deaths every system is the most frequently used classification criteria to year, breast cancer alone accounts for 14% of all cancer determine the clinical stages of cancer, predict prognosis, deaths among females [1, 2]. Although considerable im- and guide treatment strategies [5, 6]. With improvements in provements have been achieved over the past few decades health consciousness and detection tools, patients in the because of advancements in screening tools and compre- early TNM stage have become the main part of BC. How- hensive therapies for BC, cancer recurrence and distant ever, for patients with both N and M statuses negative, this 2 Journal of Oncology staging system can only predict the risk of recurrence and 2.2. Tissue Microarrays Construction. TMAs were prepared metastasis to a certain degree [7]. Furthermore, the TNM using standard procedures in collaboration with Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), as previously system mainly focuses on the tumor’s biological behaviors (tumor growth, lymph node invasion, and metastasis), and described [26]. All specimens were stained with hematoxylin insufficient attention is paid to the nontumor factors that and eosin, and the most invasive tumor areas containing affect tumor progression [8, 9]. 2erefore, it is imperative to both tumor cells and tumor stroma were identified. Cor- develop a more accurate classification system by integrating responding areas were marked in the paraffin blocks. From the new and easily available prognostic factors into the two marked areas of each paraffin block, two cores were traditional TNM staging system. taken using punch cores and deposited into the tissue Recently, accumulating evidence suggests that tumor microarray block with 70 cylinders. 2en, seven TMAs with progression and metastasis are not only affected by bio- 480 cores were constructed. Duplicates of cylinders were logical behaviors of cancer cells but also by tumor micro- included in each specimen to ensure reproducibility and environments (TMEs), which are defined as the bidirectional homogenous staining of the slides. interactions between tumor cells, stromal cells, and cellular elements [10, 11]. Tumor stroma, as an important compo- 2.3. IHC Staining of CK. IHC staining of CK was performed nent of TME, promotes tumor progression through pro- in our previous study [7]. First, TMAs were heated at 60 C duction of various nutrition, growth factors, chemokines, for 2 h, immersed in dimethylbenzene for 15 min to and cytokines [12–14]. As a result, the tumor-stromal ratio deparaffinizing, and rehydrated in a series of alcohol. 2en, (TSR), a new parameter that represents the proportion of the slides were pretreated in 0.01 mol/L citrate buffer (pH tumor-associated stroma, was introduced to the field of 6.0) and heated in a microwave oven (95 C) for 15 min. After cancer research [15]. Previous studies have demonstrated cooling at room temperature, TMAs were blocked with that the TSR is a new prognostic factor in cases of colon 0.03% hydrogen peroxide methanol for 10 min and 2% carcinoma [16], rectal adenocarcinoma [17], hepatocellular bovine serum albumin (BSA) for 20 min to decrease back- carcinoma [18], non-small cell lung cancer [19], gallbladder ground intensity. Every slide was treated overnight at 4 C cancer [20], and breast cancer [21]. Currently, the TSR is with 250 μl mouse anti-human CK monoclonal antibody largely assessed in the hematoxylin-eosin (HE) staining (AE1/AE3, dilution 1 :100, ZSGB-BIO, Beijing, China) and section, which may not accurately identify the boundary of incubated with corresponding secondary antibody (dilution tumor nests (TNs) due to low contrast between tumor and 1 : 250) for 30 min at 37 C. DAB (dilution 1 : 500, DAKO, stroma and is unsuitable for large sample detection, com- Denmark) was then added and reacted for 2 min, and the puter recognition, and automatic analysis. As a result, we use samples were counterstained with hematoxylin and sealed tissue microarrays (TMAs) containing BC specimens, which with resin mount. Furthermore, HE-stained invasive BC were stained with cytokeratin (CK) in our previous study, to sections were selected to make a comparison. specifically label tumor cells [7]. 2is study aimed at exploring the prognostic value of the TSR in invasive BC using CK-stained TMAs. Furthermore, a 2.4. Assessment of TSR. 2e TSR was defined as the ratio of new staging system combining TSR, tumor staging, lymph the tumor area to stromal area under a microscope. Prin- node staining, and metastasis is established to optimize risk ciples of the TSR scoring in this study were applied stratification for invasive BC. according to the previously described criteria [27]. Com- partments, including necrosis, microvessels, inflammation, and mucus-forming tumor tissue, were excluded. When the 2. Materials and Methods whole field of the microscope image was not filled with 2.1. Patients and Specimens. Our center has established a tumor tissue, areas that did not contain any tumor tissue clinical database of BC, which has been the data source of would also be excluded. Two researchers assessed the tumor- several clinical and translational studies [22, 23]. From the stromal ratio independently using a 10 × objective lens. 2e database, 240 invasive BC specimens were selected, and TMAs field of the highest stromal percentage from the two cores of were prepared based on a clearly set criterion. Major clini- each case were considered crucial. Disagreement on the copathologic characteristics including age, menopausal status, results was resolved by consensus. A third expert observer histological type, T stage, N status, estrogen receptor (ER) made the determination when no consensus could be status, and HER2 gene status of these patients were sum- reached. Concordance calculation and Cohen’s kappa co- marized. TNM staging and histological grading were deter- efficient were used to assess agreement between the two independent observers in categorizing the TSR as high TSR mined according to the 8th edition of the UICC/AJCC TNM classification [24] and WHO histological grading [25]. 2e or low TSR. Furthermore, the number of cases was noted, for failure event of BC patients was locoregional recurrence, which the third observer was consulted. According to metastatic recurrence, or death. 2e 5-year disease-free sur- previous studies [20, 28], a 50% cutoff point was usually vival (5-DFS) was used as the primary endpoint. Approval of selected to divide patients into stroma-low (proportion of the study protocol was obtained from the Institutional Ethics stroma <50%) and stroma-high (proportion of stroma Committee of Zhongnan Hospital of Wuhan University. 2e ≥50%) groups. As the TSR was defined as the ratio of the study was undertaken according to the ethical standards of the tumor area to stromal area under a microscope, the cutoff World Medical Association Declaration of Helsinki. point of the TSR was selected as 1 (proportion of Journal of Oncology 3 200µm 200µm (a) (b) 200µm 200µm (c) (d) Figure 1: IHC staining and HE staining results in TMAs. IHC staining of CK could specifically label tumor areas with clear contrast (a, b). 2e differentiation of tumor and stroma in HE staining was not as clear (c, d). Examples of low TSR (high stroma) (a, c); examples of high TSR (low stroma) (b, d). HE,hematoxylin-eosin; IHC, immunohistochemistry; CK, cytokeratin; TSR, tumor-stromal ratio. stroma � 50% equals to TSR � 1). TSR> 1 and TSR≤ 1 were 3. Results categorized as high TSR (low stroma) and low TSR (high 3.1. IHC Staining Results in TMAs. Typical examples of low stroma) groups, respectively. TSR (high stroma) and high TSR (low stroma) cores in IHC staining and HE staining are shown in Figure 1. Figures 1(a) 2.5. Statistical Analysis. Statistical analysis was performed and 1(c) show the staining of specimens from the same using IBM SPSS statistics (version 23.0 for Windows). 2e patient with a low TSR, and Figures 1(b) and 1(d) show the correlation between the TSR and other clinicopathologic staining of specimens from the same patient with a high factors was measured using the chi-squared test or Fisher’s TSR. After IHC staining, there was a strong color contrast of exact test. 2e Kaplan–Meier method and the log-rank test brown tumor cells and off-white tumor stroma (Figures 1(a) were performed to analyze five-year disease-free survival (5- and 1(b)). By contrast, the differentiation of tumor and DFS). Unadjusted HRs (hazard ratios) and 95% CIs (con- stroma in HE staining was not as clear as IHC staining. 2is fidence intervals) of TSR for 5-DFS in each subgroup were may not clearly reveal the edge of all tumor nests calculated using a Cox proportional hazards model. Uni- (Figures 1(c) and 1(d)). variable and multivariable survival analyses were performed by the Cox proportional hazards method. 2e proportional hazard assumption was tested based on Schoenfeld residuals. 3.2. Evaluation of Tumor-Stromal Ratio. According to the Receiver operating characteristic (ROC) curve analysis was definition of the TSR, BC patients were categorized into the applied to determine the discriminatory ability of the tumor high TSR (low stroma) and low TSR (high stroma) groups staging system. Two-sided P< 0.05 was considered statis- with 1 as the dividing value. Among 240 specimens, 52.5% tically significant. were determined as the high TSR and 47.5% as the low TSR. 4 Journal of Oncology Table 1: Relationship between TSR and major clinicopathological characteristics. Characteristics Total, n (%) Low TSR, n (%) High TSR, n (%) P value Age (years) 0.636 ≤50 149 (62.1) 69 (60.5) 80 (63.5) >50 91 (37.9) 45 (39.5) 46 (36.5) Menopausal status 0.927 Premenopausal 134 (55.8) 64 (56.1) 70 (55.6) Postmenopausal 106 (44.2) 50 (43.9) 56 (44.4) Histological type 0.044 Invasive ductal carcinoma 191 (79.6) 97 (85.1) 94 (74.6) Others 49 (20.4) 17 (14.9) 32 (25.4) T stage (cm) 0.966 T1 (T≤ 2) 35 (15.0) 17 (14.9) 18 (14.3) T2 (2< T≤ 5) 162 (67.5) 76 (66.7) 86 (68.2) T3 (T> 5) 43 (17.5) 21 (18.4) 22 (17.5) N status 0.327 N negative 109 (45.4) 48 (42.1) 61 (48.4) N positive 131 (54.6) 66 (57.9) 65 (51.6) Histological grade 0.302 I 40 (16.7) 15 (13.2) 25 (19.8) II 141 (58.8) 72 (63.2) 69 (54.8) III 59 (24.6) 27 (23.6) 32 (25.4) ER status 0.164 Positive 106 (44.2) 45 (39.5) 61 (48.4) Negative 134 (55.8) 69 (60.5) 65 (51.6) HER2 gene 0.943 Amplification 51 (21.3) 24 (21.0) 27 (21.4) Nonamplification 189 (78.7) 90 (79.0) 99 (78.6) a b ER was determined by immunohistochemistry staining according to the guideline [29]; HER2 gene was determined by fluorescent in situ hybridization (FISH) according to the guideline [30]. BC, breast cancer; T, tumor; N, node; TSR, tumor-stromal ratio; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. In n � 28 (12%) cases, no consensus could be reached and the 1.0 TSR was determined by the third observer. Concordance was 88.0% and Cohen’s kappa value was 0.77, which reflected good agreement with TSR assessment. 0.8 3.3. Correlation between TSR and Major Clinical 0.6 Characteristics. 2e prepared 7 tissue microarrays (TMAs) contained 240 invasive BC specimens. 2e age of the selected patients ranged from 29 to 78 years (median, 48 years) at the 0.4 date of surgery. Table 1 listed the major clinicopathological characteristics including age, menopausal status, histological type, T stage, N status, ER status, and HER2 gene status 0.2 χ = 5.212 grouped by tumor-stroma ratio. 2e TSR correlated with the histological type (P � 0.044) but not with age (P � 0.636), P = 0.022 menopausal status (P � 0.927), T stage (P � 0.966), N status 0.0 (P � 0.327), histological grade (P � 0.302), ER status 0.0 12.0 24.0 36.0 48.0 60.0 (P � 0.164), and HER2 gene status (P � 0.943) (Table 1). Time (months) Tumor stromal ratio 3.4. Prognosis of BC Patients according to TSR. For 240 in- High Low vasive BC patients, the 5-year disease-free survival rate was 62.0%. Traditional factors including T stage, N status, his- Figure 2: Differences between Kaplan–Meier plots for disease-free tological grade, histological type, ER status, HER2 gene survival in each group calculated by the log-rank test. Low TSR status, and menopausal status were associated with invasive (high stroma) was associated with worse 5-year disease-free sur- BC patients’ 5-DFS (P< 0.05 for all) (Supplementary vival (χ � 5.212, P � 0.022). BC � breast cancer; TS � tumor- Table 1). 2e Kaplan–Meier survival curve for high- and stromal ratio. Cumulative disease-free survival Journal of Oncology 5 Subgroup Low TSR High TSR Hazard ratio (95% CI) All patients 114 126 0.62 (0.41–0.94) Age ≤50 69 80 0.61 (0.36–1.04) >50 45 46 0.65 (0.34–1.27) Menopausal status Premenopausal 64 70 0.62 (0.34–1.11) Postmenopausal 50 56 0.63 (0.35–1.13) Histological type Inv asive ductal carcinoma 97 94 0.67 (0.43–1.05) Others 17 32 0.56 (0.19–1.67) T stage (cm) T1 (T ≤ 2) 17 18 0.94 (0.13–6.70) T2 (2 < T ≤ 5) 76 86 0.56 (0.34–0.94) T3 (T > 5) 21 22 0.68 (0.32–1.45) N status N negative 61 48 0.41 (0.15–1.10) N positive 66 65 0.74 (0.47–1.17) Histological grade I 15 25 0.60 (0.09–4.29) II 72 69 0.38 (0.20–0.75) III 27 32 0.95 (0.54–1.69) ER status Positive 45 61 0.37 (0.16–0.83) Negative 69 65 0.87 (0.54–1.41) HER2 gene Amplification 24 27 0.74 (0.36–1.54) Nonamplification 90 99 0.57 (0.34–0.94) High TSR better Low TSR better Figure 3: Forest plots of the TSR for association with 5-DFS in each subgroup. 2e dashed line showed the hazard ratio of 0.62 in all patients. CI � confidence interval. low-TSR patients are shown in Figure 2. 2e 5-DFS in high- HER2 gene nonamplification groups (HR 0.565; 95% CI TSR (low stroma) and low-TSR (high stroma) groups were 0.340–0.939; P � 0.028). Furthermore, an association be- 69.0% and 54.3%, respectively, and the difference is statis- tween high TSR and improved 5-DFS was observed in subgroups such as age, menopausal status, N status, and tically significant (χ � 5.212, P � 0.022). 2e result sug- gested that the tumor-stromal ratio may be a prognostic histological type but was not statistically significant parameter for invasive BC, and more stroma in tumor tissues (P> 0.05). HR of the T1 stage and histological grade I groups indicated worse prognosis of BC patients. had a very broad confidence interval, probably caused by the relatively small sample size or wide sample variability. 3.5. Subgroup Analysis of the TSR for Association with 5-DFS. 2e prognostic value of the TSR for 5-DFS was analyzed in 3.6. Univariable and Multivariable Analysis of the TSR and each subgroup (Figure 3). High TSR (low stroma) was as- Other Parameters. To proceed to a deeper analysis, the Cox sociated with improved 5-DFS in all patients (HR 0.621; 95% univariable and multivariable models were applied to ana- CI 0.410–0.941; P � 0.025). Subgroup analysis revealed that lyze the correlation between clinicopathological parameters the TSR was significantly associated with 5-DFS in T2 (HR and 5-DFS. Univariable analysis demonstrated that the T 0.562; 95% CI 0.336–0.940; P � 0.028), histological grade II stage (P< 0.001), N status (P< 0.001), histological grade (HR 0.383; 95% CI 0.195–0.754; P � 0.006), ER status (P< 0.001), ER status (P< 0.001), HER2 gene status positive (HR 0.366; 95% CI 0.162–0.829; P � 0.016) and (P< 0.001), and TSR (P � 0.025) were of prognostic 6 Journal of Oncology Table 2: Univariable and multivariable analysis of parameters associated with 5-DFS. Univariable analysis Multivariable analysis Parameters HR (95% CI) P value HR (95% CI) P value T stage 2.542 (1.753–3.686) <0.001 1.583 (1.100–2.280) 0.014 N status 5.035 (2.966–8.545) <0.001 3.948 (2.302–6.772) <0.001 Histological grade 4.439 (3.063–6.433) <0.001 2.825 (1.883–4.236) <0.001 ER status 0.363 (0.237–0.555) <0.001 0.567 (0.358–0.897) 0.015 HER2 gene 2.398 (1.541–3.733) <0.001 1.614 (0.995–2.618) 0.053 TSR 1.610 (1.062–2.440) 0.025 1.742 (1.137–2.669) 0.011 T, tumor; N, node; TSR, tumor-stromal ratio; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. stratify the risk of recurrence. 2e survival curve indicated Table 3: Definition of the TNM and Ts-TNM staging system. that the TNM staging system can well distinguish BC pa- TNM stage T N M TNM stage Ts Ts-TNM stage tients into three subgroups with different prognoses I T1 N0 M0 I Ts0 I 2 (χ � 59.657, P< 0.001). Similarly, the Ts-TNM staging II T0 N1 M0 Ts1 II system can also appropriately distinguish BC patients into T1 N1 M0 II Ts0 II four subgroups with different prognoses (χ � 65.041, T2 N0 M0 Ts1 III P< 0.001). 2e χ value of the TS-TNM staging system T2 N1 M0 III Ts0 III (χ � 65.041) was higher than that of the TNM staging system T3 N0 M0 Ts1 IV (χ � 59.657) (Figures 4(b) and 4(c)). III T0 N2 M0 T1 N2 M0 T2 N2 M0 T3 N1-2 M0 3.8. Comparison of the Predictive Value of the TNM and Ts- T4 N0–2 M0 TNM Staging System. A ROC curve analysis was applied to Any T N3 M0 compare the ability of the TNM and Ts-TNM staging sys- TNM, tumor node metastasis; Ts-TNM, tumor-stroma tumor node me- tems to predict recurrence. 2e AIC value and Harrell’s C tastasis; T, tumor; N, node; Ts, tumor-stroma; Ts1, low TSR (high stroma); value of the two staging systems were also calculated. 2e Ts0, high TSR (low stroma). area under the curve (AUC) of the Ts-TNM staging system (AUC: 0.727; 95% CI: 0.661–0.794) was slightly larger than the AUC of the TNM staging system (AUC: 0.723; 95%CI: significance. Multivariable analysis identified the T stage 0.657–0.790). 2e AIC value and Harrell’s C value of the (P � 0.014), N status (P< 0.001), histological grade TNM staging system were 904.308 and 0.691, respectively, (P< 0.001), ER receptor status (P � 0.015), and TSR while those of the Ts-TNM staging system were 908.425 and (P � 0.011) as independent prognostic factors (Table 2). HR 0.687, respectively. 2is demonstrates that the ability of the of the TSR was 1.742 (95% CI, 1.137–2.669), which was lower Ts-TNM staging system to predict recurrence was not lower than the N status and histological grade but higher than T than the TNM staging system’s ability. stage, ER status and HER2 gene status. 4. Discussion 3.7. Establishment and Prognostic Analysis of the Ts-TNM Staging System. To further investigate prognostic signifi- Accumulating evidence has emphasized the significance of cance of the TSR, the TSR was integrated into the TNM TME in tumor progression, and the importance of the TSR, staging system to establish a new Ts-TNM (tumor stroma as a new parameter which represents the amount of tumor- tumor-node-metastasis) staging system. As the low-TSR associated stroma, has been reported in different cancer (high stroma) group was associated with worsened disease- types. Recent studies focused largely on the prognostic value free survival, patients in the low-TSR group were defined as of the TSR, as the high percentage of tumor stroma tends to Ts1 and patients in the high-TSR group as Ts0. Patients of be correlated with unfavorable prognosis [16, 17]. the low-TSR (Ts1) group in the TNM staging system were Reliable assessment is the basis to explore prognosis of assigned to the next higher stage in the Ts-TNM staging the TSR. Currently, there are two methods to assess the TSR system, and patients of the high-TSR (Ts0) in the TNM in the HE-stained section. One is visual eyeballing, a manual staging system remained at the same stage in the Ts-TNM method with two-steps to determine the TSR [31]. First, staging system (Table 3). Based on this criterion, 9 patients of observers select the most invasive tumor areas at low the low-TSR group in TNM stage I were assigned to Ts-TNM magnification. 2en, fields containing tumor cells and tumor stage II, 65 patients of the low-TSR group in TNM stage II stroma are assessed at high magnification, and the estimate is were assigned to Ts-TNM stage III, and 38 patients of the recorded as the tumor-stromal ratio and scored per tenfold low-TSR group in stage III were assigned to Ts-TNM stage percentage. 2e other is a semiautomated point counting IV. method developed by West et al. [32], and validated for A Kaplan–Meier survival analysis was applied to verify breast cancer by Downey et al. [33]. Four-micrometer-thick the ability of the TNM and Ts-TNM staging systems to HE-stained sections are scanned, and the most invasive Journal of Oncology 7 TNM staging system Ts-TNM staging system Stage I (n = 18) Stage I (n = 9) n = 9 Stage II (n = 151) Stage II (n = 95) n = 65 Stage III (n = 71) Stage III (n = 98) n = 38 Stage III (n = 38) I I III II II IV III (a) 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 2 2 0.2 χ = 59.657 0.2 χ = 65.041 P < 0.001 P < 0.001 0.0 0.0 0.0 12.0 24.0 36.0 48.0 60.0 0.0 12.0 24.0 36.0 48.0 60.0 Time (months) Time (months) TNM stage Ts-TNM stage I I III II II IV III (b) (c) Figure 4: Patient distribution and prognostic analysis of the TNM and Ts-TNM staging system. Patients of low TSR (Ts1) in the TNM staging system were assigned to the next higher stage in the Ts-TNM staging system (red arrow), and patients of high TSR (Ts0) in the TNM staging system remained at the same stage in the Ts-TNM staging system (blue arrow) (Table 3). (a) Both TNM and Ts-TNM staging system can well distinguish BC patients into subgroups with different prognosis (b and c). TNM � tumor node metastasis; Ts-TNM � tumor-stroma tumor node metastasis; T � tumor; N � node. carcinoma cells varied in different sampling sites. As a result, tumor areas were selected. Subsequently, a total of 300 points are randomly inserted into the selected area. 2e histopa- the most invasive tumor areas containing both tumor cells thology is categorized as “tumor,” “stroma,” or “unclassified and the tumor stroma were identified and marked to (necrosis, blood vessels, and inflammation).” 2e ultimate construct TMAs. Two tumor cores were taken from each TSR is the number of points that are categorized as “tumor” specimen, and the field with the highest stromal percentage divided by the number of points that are categorized as from two cores was considered crucial. 2ese two advantages “stroma.” Scores are given per tenfold percentage. make it more objective and efficient for observers to assess 2ese two methods have been applied in various studies the TSR compared with other similar studies. More im- [33–35]. Both methods assess the TSR using HE-stained portantly, the utilization of the CK-stained TMAs may contribute to future potential large sample detections, histologic sections and could be easily implemented in routine pathology diagnostics. However, sometimes, the computer recognitions, and automatic analyses. So far, the TSR has been reported to be of prognostic boundary of the tumor nests cannot be accurately identified because of the low contrast between the tumor and stroma, value for BC in several studies. Kruijf et al. [21] demon- which may compromise the repeatability of the results and strated that early BC patients with stroma-rich tumors had a making it difficult to perform accurate recognition and higher risk of relapse than those with stroma-poor tumors, analysis. As a result, IHC staining of the CK was applied to especially in triple-negative breast cancer patients. Moor- specifically label the tumor cells in our study. It resulted in a man et al. [36] also identified the TSR as a strong inde- strong color contrast of marking tumor cells in brown and pendent prognostic variable in triple-negative breast cancer the tumor stroma in off-white. Moreover, it is well known patients. A study from the perioperative chemotherapy trial that breast cancer is a heterogeneous disease. Distribution of (POP trial, 10854) validates the prognostic value of the TSR Cumulative disease-free survival Cumulative disease-free survival 8 Journal of Oncology results. A computer recognition and analysis software will in lymph node-negative premenopausal BC patients [34]. 2e prognostic value of the TSR in primary operable invasive greatly enhance efficiency, which may be a more optimal method for analyzing histological images. ductal BC [35], estrogen receptor-positive BC [33], and inflammatory BC [37] was also confirmed. 2is study aimed at exploring the prognostic value of the TSR in invasive BC 5. Conclusion using CK-stained TMAs, and 240 invasive BC specimens In general, our study uses CK-stained TMAs and demon- were selected from an established clinical database. 2e strates that invasive BC patients of low TSR have poor characteristics of the patients in this study displayed similar prognoses. Furthermore, the Ts-TNM staging system features from central Chinese but is different from the combining the TSR, tumor staging, lymph node status, and general population of invasive breast cancer patients, in that metastasis staging can provide supplementary information the study population is younger in onset age and has less in predicting the risk of recurrence and metastasis and may patients with lymph node negative, small tumor size, and ER serve as a new paradigm to encompass tumor heterogeneity. positive [38, 39]. 2e lower rate of lymph node negative indicated more patients with aggressive BC, and the lower Data Availability rate of ER positive indicated more patients disqualified for endocrine therapy. As a result, prognosis of the subjects is Previously reported data on major clinicopathologic char- poorer than that of common patients with invasive breast acteristics of patients were used to support this study and are cancer. In line with previous studies, this study revealed that available at 10.1016/j.biomaterials.2010.07.091. 2ese prior invasive BC patients in the low-TSR group had a worse 5- studies (and datasets) are cited at relevant places within the DFS compared with patients in the high-TSR group, and the text as references. TSR was not associated with age, menopausal status, T stage, N status, histological grade, ER status, and HER2 gene status. Ethical Approval Subgroup analysis revealed that the TSR was significantly associated with 5-DFS in T2, histological grade II, ER status Approval of the study protocol was obtained from the In- positive, and HER2 gene status nonamplification groups. stitutional Ethics Committee of Zhongnan Hospital of Multivariable analysis identified the T stage, N status, his- Wuhan University. 2e study was undertaken according to tological grade, hormone receptor status, and TSR as in- the ethical standards of the World Medical Association dependent prognostic factors of invasive BC patients. Declaration of Helsinki. When the N and M statuses of patients is both negative, the TSR can provide information to predict the risk of re- Conflicts of Interest currence and metastasis. As a result, we integrated the TSR into the traditional TNM staging system and established a 2e authors declare that they have no conflicts of interest. new Ts-TNM staging system creatively in BC. It stratified 240 invasive BC patients into four subgroups (stages I, II, III, Authors’ Contributions and IV) with different prognosis. 2e ROC analysis dem- onstrated that the ability of the Ts-TNM staging system to Qian Xu and Jing-Ping Yuan contributed equally to this predict recurrence was not lower than the TNM staging work. system. Furthermore, the Ts-TNM staging system, com- bining tumor’s biological behaviors (tumor size, lymph node Acknowledgments spread, and distant metastasis) with stromal status (low TSR 2is work was supported by the Young Scientists Fund of and high TSR), may be a new paradigm to encompass tumor heterogeneity. Furthermore, the TSR can be easily assessed National Natural Science Foundation (grant numbers 81701768 and 81702901) and Funding for Scientific and in routine pathology diagnostics, which makes it feasible to perform Ts-TNM staging. Technological Innovation of Zhongnan Hospital of Wuhan University (znpy2016003). However, there are still some disadvantages and limi- tations in our study. First, this research is retrospective, and the sample capacity is relatively small, especially for the Supplementary Materials TNM stage I group. All 9 patients in the Ts-TNM stage I Supplementary Table 1: analysis of characteristics regarding group did not have a recurrence after 5 years, which indi- 5-DFS. (Supplementary Materials) cates that the prognostic value of the TSR for BC patients with TNM stage I remains uncertain. It will be valuable to References conduct a prospective study with a larger sample for the TNM stage I group. Second, although TMAs are strictly [1] L. A. Torre, F. Bray, R. L. Siegel, J. Ferlay, J. Lortet-Tieulent, constructed according to the criterion that only the most and A. 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Prognostic Significance of the Tumor-Stromal Ratio in Invasive Breast Cancer and a Proposal of a New Ts-TNM Staging System

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Copyright © 2020 Qian Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Journal of Oncology Volume 2020, Article ID 9050631, 10 pages https://doi.org/10.1155/2020/9050631 Research Article Prognostic Significance of the Tumor-Stromal Ratio in Invasive Breast Cancer and a Proposal of a New Ts-TNM Staging System 1,2 3 1,4 1,4 Qian Xu , Jing-Ping Yuan, Yuan-Yuan Chen, Hong-Yan Zhang, 1,4 1,2 Lin-Wei Wang , and Bin Xiong Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Zhongnan Hospital of Wuhan University, 4300711 Wuhan, China Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China Department of Pathology, Renmin Hospital of Wuhan University, 430060 Wuhan, China Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan, China Correspondence should be addressed to Lin-Wei Wang; wanglinweiwhu@163.com and Bin Xiong; binxiong1961@whu.edu.cn Received 7 October 2019; Revised 16 January 2020; Accepted 5 February 2020; Published 21 April 2020 Academic Editor: Vincenzo Coppola Copyright © 2020 Qian Xu et al. 2is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Previous studies have demonstrated that the tumor-stromal ratio (TSR) was an independent prognostic factor in several types of carcinomas. 2is study aimed at exploring the prognostic significance of the TSR in invasive breast cancer using immunohistochemistry (IHC)-stained tissue microarrays (TMAs) and integrating the TSR into the traditional tumor-node- metastasis (TNM) staging system. Methods. 2e prepared 7 TMAs containing 240 patients with 480 invasive BC specimens were stained with cytokeratin (CK) by the IHC staining method. 2e ratio of tumor cells and stromal cells was visually assessed. TSR > 1 and TSR≤ 1 were categorized as the high TSR (low stroma) and low TSR (high stroma) groups, respectively, and the prognostic value of the TSR at 5-year disease-free survival (5-DFS) was analyzed. A new Ts-TNM (tumor stroma-tumor-node-metastasis) staging system was established and assessed. Results. IHC staining of CK could specifically label tumor cells with clear contrast, making it easy to manually assess TSR. High TSR (low stroma) and low TSR (high stroma) were observed in 52.5% (n � 126) and 47.5 (n � 114) of the cases, according to the division of value 1. A Kaplan–Meier analysis showed that patients in the low TSR group had a worse 5-DFS compared with patients in the high TSR group (P � 0.022). Multivariable analysis indicated that the T stage (P � 0.014), N status (P< 0.001), histological grade (P< 0.001), estrogen receptor status (P � 0.015), and TSR (P � 0.011) were independent prognostic factors of invasive BC patients. 2e new Ts-TNM staging system combining TSR, tumor staging, lymph node status, and metastasis staging was established. 2e receiver operating characteristic (ROC) curve analysis dem- onstrated that the ability of the Ts-TNM staging system to predict recurrence was not lower than that of the TNM staging system. Conclusions. 2is study confirms that the TSR is a prognostic indicator for invasive breast cancer. 2e Ts-TNM staging system containing stromal and tumor information may optimize risk stratification for invasive breast cancer. metastasis still occurs in a proportion of patients [3, 4]. More 1. Introduction prognostic factors are needed to optimize invasive breast Breast cancer (BC) is the leading cause of cancer deaths cancer risk stratification to guide precise treatment. among females in both developed and developing countries. Currently, the tumor-node-metastasis (TNM) staging With an estimated 1.6 million cases and 520,000 deaths every system is the most frequently used classification criteria to year, breast cancer alone accounts for 14% of all cancer determine the clinical stages of cancer, predict prognosis, deaths among females [1, 2]. Although considerable im- and guide treatment strategies [5, 6]. With improvements in provements have been achieved over the past few decades health consciousness and detection tools, patients in the because of advancements in screening tools and compre- early TNM stage have become the main part of BC. How- hensive therapies for BC, cancer recurrence and distant ever, for patients with both N and M statuses negative, this 2 Journal of Oncology staging system can only predict the risk of recurrence and 2.2. Tissue Microarrays Construction. TMAs were prepared metastasis to a certain degree [7]. Furthermore, the TNM using standard procedures in collaboration with Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), as previously system mainly focuses on the tumor’s biological behaviors (tumor growth, lymph node invasion, and metastasis), and described [26]. All specimens were stained with hematoxylin insufficient attention is paid to the nontumor factors that and eosin, and the most invasive tumor areas containing affect tumor progression [8, 9]. 2erefore, it is imperative to both tumor cells and tumor stroma were identified. Cor- develop a more accurate classification system by integrating responding areas were marked in the paraffin blocks. From the new and easily available prognostic factors into the two marked areas of each paraffin block, two cores were traditional TNM staging system. taken using punch cores and deposited into the tissue Recently, accumulating evidence suggests that tumor microarray block with 70 cylinders. 2en, seven TMAs with progression and metastasis are not only affected by bio- 480 cores were constructed. Duplicates of cylinders were logical behaviors of cancer cells but also by tumor micro- included in each specimen to ensure reproducibility and environments (TMEs), which are defined as the bidirectional homogenous staining of the slides. interactions between tumor cells, stromal cells, and cellular elements [10, 11]. Tumor stroma, as an important compo- 2.3. IHC Staining of CK. IHC staining of CK was performed nent of TME, promotes tumor progression through pro- in our previous study [7]. First, TMAs were heated at 60 C duction of various nutrition, growth factors, chemokines, for 2 h, immersed in dimethylbenzene for 15 min to and cytokines [12–14]. As a result, the tumor-stromal ratio deparaffinizing, and rehydrated in a series of alcohol. 2en, (TSR), a new parameter that represents the proportion of the slides were pretreated in 0.01 mol/L citrate buffer (pH tumor-associated stroma, was introduced to the field of 6.0) and heated in a microwave oven (95 C) for 15 min. After cancer research [15]. Previous studies have demonstrated cooling at room temperature, TMAs were blocked with that the TSR is a new prognostic factor in cases of colon 0.03% hydrogen peroxide methanol for 10 min and 2% carcinoma [16], rectal adenocarcinoma [17], hepatocellular bovine serum albumin (BSA) for 20 min to decrease back- carcinoma [18], non-small cell lung cancer [19], gallbladder ground intensity. Every slide was treated overnight at 4 C cancer [20], and breast cancer [21]. Currently, the TSR is with 250 μl mouse anti-human CK monoclonal antibody largely assessed in the hematoxylin-eosin (HE) staining (AE1/AE3, dilution 1 :100, ZSGB-BIO, Beijing, China) and section, which may not accurately identify the boundary of incubated with corresponding secondary antibody (dilution tumor nests (TNs) due to low contrast between tumor and 1 : 250) for 30 min at 37 C. DAB (dilution 1 : 500, DAKO, stroma and is unsuitable for large sample detection, com- Denmark) was then added and reacted for 2 min, and the puter recognition, and automatic analysis. As a result, we use samples were counterstained with hematoxylin and sealed tissue microarrays (TMAs) containing BC specimens, which with resin mount. Furthermore, HE-stained invasive BC were stained with cytokeratin (CK) in our previous study, to sections were selected to make a comparison. specifically label tumor cells [7]. 2is study aimed at exploring the prognostic value of the TSR in invasive BC using CK-stained TMAs. Furthermore, a 2.4. Assessment of TSR. 2e TSR was defined as the ratio of new staging system combining TSR, tumor staging, lymph the tumor area to stromal area under a microscope. Prin- node staining, and metastasis is established to optimize risk ciples of the TSR scoring in this study were applied stratification for invasive BC. according to the previously described criteria [27]. Com- partments, including necrosis, microvessels, inflammation, and mucus-forming tumor tissue, were excluded. When the 2. Materials and Methods whole field of the microscope image was not filled with 2.1. Patients and Specimens. Our center has established a tumor tissue, areas that did not contain any tumor tissue clinical database of BC, which has been the data source of would also be excluded. Two researchers assessed the tumor- several clinical and translational studies [22, 23]. From the stromal ratio independently using a 10 × objective lens. 2e database, 240 invasive BC specimens were selected, and TMAs field of the highest stromal percentage from the two cores of were prepared based on a clearly set criterion. Major clini- each case were considered crucial. Disagreement on the copathologic characteristics including age, menopausal status, results was resolved by consensus. A third expert observer histological type, T stage, N status, estrogen receptor (ER) made the determination when no consensus could be status, and HER2 gene status of these patients were sum- reached. Concordance calculation and Cohen’s kappa co- marized. TNM staging and histological grading were deter- efficient were used to assess agreement between the two independent observers in categorizing the TSR as high TSR mined according to the 8th edition of the UICC/AJCC TNM classification [24] and WHO histological grading [25]. 2e or low TSR. Furthermore, the number of cases was noted, for failure event of BC patients was locoregional recurrence, which the third observer was consulted. According to metastatic recurrence, or death. 2e 5-year disease-free sur- previous studies [20, 28], a 50% cutoff point was usually vival (5-DFS) was used as the primary endpoint. Approval of selected to divide patients into stroma-low (proportion of the study protocol was obtained from the Institutional Ethics stroma <50%) and stroma-high (proportion of stroma Committee of Zhongnan Hospital of Wuhan University. 2e ≥50%) groups. As the TSR was defined as the ratio of the study was undertaken according to the ethical standards of the tumor area to stromal area under a microscope, the cutoff World Medical Association Declaration of Helsinki. point of the TSR was selected as 1 (proportion of Journal of Oncology 3 200µm 200µm (a) (b) 200µm 200µm (c) (d) Figure 1: IHC staining and HE staining results in TMAs. IHC staining of CK could specifically label tumor areas with clear contrast (a, b). 2e differentiation of tumor and stroma in HE staining was not as clear (c, d). Examples of low TSR (high stroma) (a, c); examples of high TSR (low stroma) (b, d). HE,hematoxylin-eosin; IHC, immunohistochemistry; CK, cytokeratin; TSR, tumor-stromal ratio. stroma � 50% equals to TSR � 1). TSR> 1 and TSR≤ 1 were 3. Results categorized as high TSR (low stroma) and low TSR (high 3.1. IHC Staining Results in TMAs. Typical examples of low stroma) groups, respectively. TSR (high stroma) and high TSR (low stroma) cores in IHC staining and HE staining are shown in Figure 1. Figures 1(a) 2.5. Statistical Analysis. Statistical analysis was performed and 1(c) show the staining of specimens from the same using IBM SPSS statistics (version 23.0 for Windows). 2e patient with a low TSR, and Figures 1(b) and 1(d) show the correlation between the TSR and other clinicopathologic staining of specimens from the same patient with a high factors was measured using the chi-squared test or Fisher’s TSR. After IHC staining, there was a strong color contrast of exact test. 2e Kaplan–Meier method and the log-rank test brown tumor cells and off-white tumor stroma (Figures 1(a) were performed to analyze five-year disease-free survival (5- and 1(b)). By contrast, the differentiation of tumor and DFS). Unadjusted HRs (hazard ratios) and 95% CIs (con- stroma in HE staining was not as clear as IHC staining. 2is fidence intervals) of TSR for 5-DFS in each subgroup were may not clearly reveal the edge of all tumor nests calculated using a Cox proportional hazards model. Uni- (Figures 1(c) and 1(d)). variable and multivariable survival analyses were performed by the Cox proportional hazards method. 2e proportional hazard assumption was tested based on Schoenfeld residuals. 3.2. Evaluation of Tumor-Stromal Ratio. According to the Receiver operating characteristic (ROC) curve analysis was definition of the TSR, BC patients were categorized into the applied to determine the discriminatory ability of the tumor high TSR (low stroma) and low TSR (high stroma) groups staging system. Two-sided P< 0.05 was considered statis- with 1 as the dividing value. Among 240 specimens, 52.5% tically significant. were determined as the high TSR and 47.5% as the low TSR. 4 Journal of Oncology Table 1: Relationship between TSR and major clinicopathological characteristics. Characteristics Total, n (%) Low TSR, n (%) High TSR, n (%) P value Age (years) 0.636 ≤50 149 (62.1) 69 (60.5) 80 (63.5) >50 91 (37.9) 45 (39.5) 46 (36.5) Menopausal status 0.927 Premenopausal 134 (55.8) 64 (56.1) 70 (55.6) Postmenopausal 106 (44.2) 50 (43.9) 56 (44.4) Histological type 0.044 Invasive ductal carcinoma 191 (79.6) 97 (85.1) 94 (74.6) Others 49 (20.4) 17 (14.9) 32 (25.4) T stage (cm) 0.966 T1 (T≤ 2) 35 (15.0) 17 (14.9) 18 (14.3) T2 (2< T≤ 5) 162 (67.5) 76 (66.7) 86 (68.2) T3 (T> 5) 43 (17.5) 21 (18.4) 22 (17.5) N status 0.327 N negative 109 (45.4) 48 (42.1) 61 (48.4) N positive 131 (54.6) 66 (57.9) 65 (51.6) Histological grade 0.302 I 40 (16.7) 15 (13.2) 25 (19.8) II 141 (58.8) 72 (63.2) 69 (54.8) III 59 (24.6) 27 (23.6) 32 (25.4) ER status 0.164 Positive 106 (44.2) 45 (39.5) 61 (48.4) Negative 134 (55.8) 69 (60.5) 65 (51.6) HER2 gene 0.943 Amplification 51 (21.3) 24 (21.0) 27 (21.4) Nonamplification 189 (78.7) 90 (79.0) 99 (78.6) a b ER was determined by immunohistochemistry staining according to the guideline [29]; HER2 gene was determined by fluorescent in situ hybridization (FISH) according to the guideline [30]. BC, breast cancer; T, tumor; N, node; TSR, tumor-stromal ratio; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. In n � 28 (12%) cases, no consensus could be reached and the 1.0 TSR was determined by the third observer. Concordance was 88.0% and Cohen’s kappa value was 0.77, which reflected good agreement with TSR assessment. 0.8 3.3. Correlation between TSR and Major Clinical 0.6 Characteristics. 2e prepared 7 tissue microarrays (TMAs) contained 240 invasive BC specimens. 2e age of the selected patients ranged from 29 to 78 years (median, 48 years) at the 0.4 date of surgery. Table 1 listed the major clinicopathological characteristics including age, menopausal status, histological type, T stage, N status, ER status, and HER2 gene status 0.2 χ = 5.212 grouped by tumor-stroma ratio. 2e TSR correlated with the histological type (P � 0.044) but not with age (P � 0.636), P = 0.022 menopausal status (P � 0.927), T stage (P � 0.966), N status 0.0 (P � 0.327), histological grade (P � 0.302), ER status 0.0 12.0 24.0 36.0 48.0 60.0 (P � 0.164), and HER2 gene status (P � 0.943) (Table 1). Time (months) Tumor stromal ratio 3.4. Prognosis of BC Patients according to TSR. For 240 in- High Low vasive BC patients, the 5-year disease-free survival rate was 62.0%. Traditional factors including T stage, N status, his- Figure 2: Differences between Kaplan–Meier plots for disease-free tological grade, histological type, ER status, HER2 gene survival in each group calculated by the log-rank test. Low TSR status, and menopausal status were associated with invasive (high stroma) was associated with worse 5-year disease-free sur- BC patients’ 5-DFS (P< 0.05 for all) (Supplementary vival (χ � 5.212, P � 0.022). BC � breast cancer; TS � tumor- Table 1). 2e Kaplan–Meier survival curve for high- and stromal ratio. Cumulative disease-free survival Journal of Oncology 5 Subgroup Low TSR High TSR Hazard ratio (95% CI) All patients 114 126 0.62 (0.41–0.94) Age ≤50 69 80 0.61 (0.36–1.04) >50 45 46 0.65 (0.34–1.27) Menopausal status Premenopausal 64 70 0.62 (0.34–1.11) Postmenopausal 50 56 0.63 (0.35–1.13) Histological type Inv asive ductal carcinoma 97 94 0.67 (0.43–1.05) Others 17 32 0.56 (0.19–1.67) T stage (cm) T1 (T ≤ 2) 17 18 0.94 (0.13–6.70) T2 (2 < T ≤ 5) 76 86 0.56 (0.34–0.94) T3 (T > 5) 21 22 0.68 (0.32–1.45) N status N negative 61 48 0.41 (0.15–1.10) N positive 66 65 0.74 (0.47–1.17) Histological grade I 15 25 0.60 (0.09–4.29) II 72 69 0.38 (0.20–0.75) III 27 32 0.95 (0.54–1.69) ER status Positive 45 61 0.37 (0.16–0.83) Negative 69 65 0.87 (0.54–1.41) HER2 gene Amplification 24 27 0.74 (0.36–1.54) Nonamplification 90 99 0.57 (0.34–0.94) High TSR better Low TSR better Figure 3: Forest plots of the TSR for association with 5-DFS in each subgroup. 2e dashed line showed the hazard ratio of 0.62 in all patients. CI � confidence interval. low-TSR patients are shown in Figure 2. 2e 5-DFS in high- HER2 gene nonamplification groups (HR 0.565; 95% CI TSR (low stroma) and low-TSR (high stroma) groups were 0.340–0.939; P � 0.028). Furthermore, an association be- 69.0% and 54.3%, respectively, and the difference is statis- tween high TSR and improved 5-DFS was observed in subgroups such as age, menopausal status, N status, and tically significant (χ � 5.212, P � 0.022). 2e result sug- gested that the tumor-stromal ratio may be a prognostic histological type but was not statistically significant parameter for invasive BC, and more stroma in tumor tissues (P> 0.05). HR of the T1 stage and histological grade I groups indicated worse prognosis of BC patients. had a very broad confidence interval, probably caused by the relatively small sample size or wide sample variability. 3.5. Subgroup Analysis of the TSR for Association with 5-DFS. 2e prognostic value of the TSR for 5-DFS was analyzed in 3.6. Univariable and Multivariable Analysis of the TSR and each subgroup (Figure 3). High TSR (low stroma) was as- Other Parameters. To proceed to a deeper analysis, the Cox sociated with improved 5-DFS in all patients (HR 0.621; 95% univariable and multivariable models were applied to ana- CI 0.410–0.941; P � 0.025). Subgroup analysis revealed that lyze the correlation between clinicopathological parameters the TSR was significantly associated with 5-DFS in T2 (HR and 5-DFS. Univariable analysis demonstrated that the T 0.562; 95% CI 0.336–0.940; P � 0.028), histological grade II stage (P< 0.001), N status (P< 0.001), histological grade (HR 0.383; 95% CI 0.195–0.754; P � 0.006), ER status (P< 0.001), ER status (P< 0.001), HER2 gene status positive (HR 0.366; 95% CI 0.162–0.829; P � 0.016) and (P< 0.001), and TSR (P � 0.025) were of prognostic 6 Journal of Oncology Table 2: Univariable and multivariable analysis of parameters associated with 5-DFS. Univariable analysis Multivariable analysis Parameters HR (95% CI) P value HR (95% CI) P value T stage 2.542 (1.753–3.686) <0.001 1.583 (1.100–2.280) 0.014 N status 5.035 (2.966–8.545) <0.001 3.948 (2.302–6.772) <0.001 Histological grade 4.439 (3.063–6.433) <0.001 2.825 (1.883–4.236) <0.001 ER status 0.363 (0.237–0.555) <0.001 0.567 (0.358–0.897) 0.015 HER2 gene 2.398 (1.541–3.733) <0.001 1.614 (0.995–2.618) 0.053 TSR 1.610 (1.062–2.440) 0.025 1.742 (1.137–2.669) 0.011 T, tumor; N, node; TSR, tumor-stromal ratio; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. stratify the risk of recurrence. 2e survival curve indicated Table 3: Definition of the TNM and Ts-TNM staging system. that the TNM staging system can well distinguish BC pa- TNM stage T N M TNM stage Ts Ts-TNM stage tients into three subgroups with different prognoses I T1 N0 M0 I Ts0 I 2 (χ � 59.657, P< 0.001). Similarly, the Ts-TNM staging II T0 N1 M0 Ts1 II system can also appropriately distinguish BC patients into T1 N1 M0 II Ts0 II four subgroups with different prognoses (χ � 65.041, T2 N0 M0 Ts1 III P< 0.001). 2e χ value of the TS-TNM staging system T2 N1 M0 III Ts0 III (χ � 65.041) was higher than that of the TNM staging system T3 N0 M0 Ts1 IV (χ � 59.657) (Figures 4(b) and 4(c)). III T0 N2 M0 T1 N2 M0 T2 N2 M0 T3 N1-2 M0 3.8. Comparison of the Predictive Value of the TNM and Ts- T4 N0–2 M0 TNM Staging System. A ROC curve analysis was applied to Any T N3 M0 compare the ability of the TNM and Ts-TNM staging sys- TNM, tumor node metastasis; Ts-TNM, tumor-stroma tumor node me- tems to predict recurrence. 2e AIC value and Harrell’s C tastasis; T, tumor; N, node; Ts, tumor-stroma; Ts1, low TSR (high stroma); value of the two staging systems were also calculated. 2e Ts0, high TSR (low stroma). area under the curve (AUC) of the Ts-TNM staging system (AUC: 0.727; 95% CI: 0.661–0.794) was slightly larger than the AUC of the TNM staging system (AUC: 0.723; 95%CI: significance. Multivariable analysis identified the T stage 0.657–0.790). 2e AIC value and Harrell’s C value of the (P � 0.014), N status (P< 0.001), histological grade TNM staging system were 904.308 and 0.691, respectively, (P< 0.001), ER receptor status (P � 0.015), and TSR while those of the Ts-TNM staging system were 908.425 and (P � 0.011) as independent prognostic factors (Table 2). HR 0.687, respectively. 2is demonstrates that the ability of the of the TSR was 1.742 (95% CI, 1.137–2.669), which was lower Ts-TNM staging system to predict recurrence was not lower than the N status and histological grade but higher than T than the TNM staging system’s ability. stage, ER status and HER2 gene status. 4. Discussion 3.7. Establishment and Prognostic Analysis of the Ts-TNM Staging System. To further investigate prognostic signifi- Accumulating evidence has emphasized the significance of cance of the TSR, the TSR was integrated into the TNM TME in tumor progression, and the importance of the TSR, staging system to establish a new Ts-TNM (tumor stroma as a new parameter which represents the amount of tumor- tumor-node-metastasis) staging system. As the low-TSR associated stroma, has been reported in different cancer (high stroma) group was associated with worsened disease- types. Recent studies focused largely on the prognostic value free survival, patients in the low-TSR group were defined as of the TSR, as the high percentage of tumor stroma tends to Ts1 and patients in the high-TSR group as Ts0. Patients of be correlated with unfavorable prognosis [16, 17]. the low-TSR (Ts1) group in the TNM staging system were Reliable assessment is the basis to explore prognosis of assigned to the next higher stage in the Ts-TNM staging the TSR. Currently, there are two methods to assess the TSR system, and patients of the high-TSR (Ts0) in the TNM in the HE-stained section. One is visual eyeballing, a manual staging system remained at the same stage in the Ts-TNM method with two-steps to determine the TSR [31]. First, staging system (Table 3). Based on this criterion, 9 patients of observers select the most invasive tumor areas at low the low-TSR group in TNM stage I were assigned to Ts-TNM magnification. 2en, fields containing tumor cells and tumor stage II, 65 patients of the low-TSR group in TNM stage II stroma are assessed at high magnification, and the estimate is were assigned to Ts-TNM stage III, and 38 patients of the recorded as the tumor-stromal ratio and scored per tenfold low-TSR group in stage III were assigned to Ts-TNM stage percentage. 2e other is a semiautomated point counting IV. method developed by West et al. [32], and validated for A Kaplan–Meier survival analysis was applied to verify breast cancer by Downey et al. [33]. Four-micrometer-thick the ability of the TNM and Ts-TNM staging systems to HE-stained sections are scanned, and the most invasive Journal of Oncology 7 TNM staging system Ts-TNM staging system Stage I (n = 18) Stage I (n = 9) n = 9 Stage II (n = 151) Stage II (n = 95) n = 65 Stage III (n = 71) Stage III (n = 98) n = 38 Stage III (n = 38) I I III II II IV III (a) 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 2 2 0.2 χ = 59.657 0.2 χ = 65.041 P < 0.001 P < 0.001 0.0 0.0 0.0 12.0 24.0 36.0 48.0 60.0 0.0 12.0 24.0 36.0 48.0 60.0 Time (months) Time (months) TNM stage Ts-TNM stage I I III II II IV III (b) (c) Figure 4: Patient distribution and prognostic analysis of the TNM and Ts-TNM staging system. Patients of low TSR (Ts1) in the TNM staging system were assigned to the next higher stage in the Ts-TNM staging system (red arrow), and patients of high TSR (Ts0) in the TNM staging system remained at the same stage in the Ts-TNM staging system (blue arrow) (Table 3). (a) Both TNM and Ts-TNM staging system can well distinguish BC patients into subgroups with different prognosis (b and c). TNM � tumor node metastasis; Ts-TNM � tumor-stroma tumor node metastasis; T � tumor; N � node. carcinoma cells varied in different sampling sites. As a result, tumor areas were selected. Subsequently, a total of 300 points are randomly inserted into the selected area. 2e histopa- the most invasive tumor areas containing both tumor cells thology is categorized as “tumor,” “stroma,” or “unclassified and the tumor stroma were identified and marked to (necrosis, blood vessels, and inflammation).” 2e ultimate construct TMAs. Two tumor cores were taken from each TSR is the number of points that are categorized as “tumor” specimen, and the field with the highest stromal percentage divided by the number of points that are categorized as from two cores was considered crucial. 2ese two advantages “stroma.” Scores are given per tenfold percentage. make it more objective and efficient for observers to assess 2ese two methods have been applied in various studies the TSR compared with other similar studies. More im- [33–35]. Both methods assess the TSR using HE-stained portantly, the utilization of the CK-stained TMAs may contribute to future potential large sample detections, histologic sections and could be easily implemented in routine pathology diagnostics. However, sometimes, the computer recognitions, and automatic analyses. So far, the TSR has been reported to be of prognostic boundary of the tumor nests cannot be accurately identified because of the low contrast between the tumor and stroma, value for BC in several studies. Kruijf et al. [21] demon- which may compromise the repeatability of the results and strated that early BC patients with stroma-rich tumors had a making it difficult to perform accurate recognition and higher risk of relapse than those with stroma-poor tumors, analysis. As a result, IHC staining of the CK was applied to especially in triple-negative breast cancer patients. Moor- specifically label the tumor cells in our study. It resulted in a man et al. [36] also identified the TSR as a strong inde- strong color contrast of marking tumor cells in brown and pendent prognostic variable in triple-negative breast cancer the tumor stroma in off-white. Moreover, it is well known patients. A study from the perioperative chemotherapy trial that breast cancer is a heterogeneous disease. Distribution of (POP trial, 10854) validates the prognostic value of the TSR Cumulative disease-free survival Cumulative disease-free survival 8 Journal of Oncology results. A computer recognition and analysis software will in lymph node-negative premenopausal BC patients [34]. 2e prognostic value of the TSR in primary operable invasive greatly enhance efficiency, which may be a more optimal method for analyzing histological images. ductal BC [35], estrogen receptor-positive BC [33], and inflammatory BC [37] was also confirmed. 2is study aimed at exploring the prognostic value of the TSR in invasive BC 5. Conclusion using CK-stained TMAs, and 240 invasive BC specimens In general, our study uses CK-stained TMAs and demon- were selected from an established clinical database. 2e strates that invasive BC patients of low TSR have poor characteristics of the patients in this study displayed similar prognoses. Furthermore, the Ts-TNM staging system features from central Chinese but is different from the combining the TSR, tumor staging, lymph node status, and general population of invasive breast cancer patients, in that metastasis staging can provide supplementary information the study population is younger in onset age and has less in predicting the risk of recurrence and metastasis and may patients with lymph node negative, small tumor size, and ER serve as a new paradigm to encompass tumor heterogeneity. positive [38, 39]. 2e lower rate of lymph node negative indicated more patients with aggressive BC, and the lower Data Availability rate of ER positive indicated more patients disqualified for endocrine therapy. As a result, prognosis of the subjects is Previously reported data on major clinicopathologic char- poorer than that of common patients with invasive breast acteristics of patients were used to support this study and are cancer. In line with previous studies, this study revealed that available at 10.1016/j.biomaterials.2010.07.091. 2ese prior invasive BC patients in the low-TSR group had a worse 5- studies (and datasets) are cited at relevant places within the DFS compared with patients in the high-TSR group, and the text as references. TSR was not associated with age, menopausal status, T stage, N status, histological grade, ER status, and HER2 gene status. Ethical Approval Subgroup analysis revealed that the TSR was significantly associated with 5-DFS in T2, histological grade II, ER status Approval of the study protocol was obtained from the In- positive, and HER2 gene status nonamplification groups. stitutional Ethics Committee of Zhongnan Hospital of Multivariable analysis identified the T stage, N status, his- Wuhan University. 2e study was undertaken according to tological grade, hormone receptor status, and TSR as in- the ethical standards of the World Medical Association dependent prognostic factors of invasive BC patients. Declaration of Helsinki. When the N and M statuses of patients is both negative, the TSR can provide information to predict the risk of re- Conflicts of Interest currence and metastasis. As a result, we integrated the TSR into the traditional TNM staging system and established a 2e authors declare that they have no conflicts of interest. new Ts-TNM staging system creatively in BC. It stratified 240 invasive BC patients into four subgroups (stages I, II, III, Authors’ Contributions and IV) with different prognosis. 2e ROC analysis dem- onstrated that the ability of the Ts-TNM staging system to Qian Xu and Jing-Ping Yuan contributed equally to this predict recurrence was not lower than the TNM staging work. system. Furthermore, the Ts-TNM staging system, com- bining tumor’s biological behaviors (tumor size, lymph node Acknowledgments spread, and distant metastasis) with stromal status (low TSR 2is work was supported by the Young Scientists Fund of and high TSR), may be a new paradigm to encompass tumor heterogeneity. Furthermore, the TSR can be easily assessed National Natural Science Foundation (grant numbers 81701768 and 81702901) and Funding for Scientific and in routine pathology diagnostics, which makes it feasible to perform Ts-TNM staging. Technological Innovation of Zhongnan Hospital of Wuhan University (znpy2016003). 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Published: Apr 21, 2020

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