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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The purpose of this study was to determine whether p53 protein expression in tumor stromal fibroblasts assessed immunohistochemically by the Allred score system is significantly associated with nodal metastasis by invasive ductal carcinoma (IDC), and significantly associated with the outcome of 1042 IDC patients according to adjuvant therapy status, UICC pTNM stage, and triple-negative IDC status, in multivariate analyses with well-known clinicopathological factors. The Allred scores for p53 expression in tumor stromal fibroblasts were significantly associated with the number of nodal metastases, and Allred scores of 4–8 for p53 in tumor stromal fibroblasts significantly increased the hazard rate for distant organ metastasis or for tumor death in the triple-negative IDC patients, and the UICC pTNM stage I, II, and III patients. The results indicated that p53 protein expression in tumor stromal fibroblasts is closely associated with the number of nodal metastases and the outcome of IDC patients. (Cancer Sci 2009; 00: 000–000)

It has recently been reported that the gene expression and protein expression profiles of the tumor stroma play very important roles in tumor progression in carcinoma,(1–3) and the interaction between tumor and stromal cells also plays a very important role in tumor progression by carcinoma.(4–6) We and others have already reported that a characteristic histological feature of tumor stroma, a fibrotic focus, is a very useful prognostic histological tumor stromal indicator for accurately predicting the outcome of patients with invasive ductal carcinoma (IDC),(7–10) and that growth factors produced by tumor cells and tumor stromal cells play a very important role in tumor progression by IDC.(11) In addition, proliferative activity of tumor stromal fibroblasts plays a very important role in nodal metastasis and distant organ metastasis by IDC.(12,13) These findings strongly suggest a significant role of the tumor stroma in tumor progression by IDC.

p53 is the most commonly mutated gene in human neoplasms,(14) and the p53 tumor suppressor protein is involved in the cell cycle, checkpoint control, repair of DNA damage, and apoptosis.(15,16) Also, besides their well-studied cell-autonomous role in cancer cells, mutations of the p53 tumor suppressor gene have been described in stromal fibroblasts of breast and prostate carcinoma in humans and experimental animals.(17–20) A high frequency of p53 mutations in tumor cells and the surrounding stroma has also reported,(17) and p53 mutations in breast cancer stromal cells have been reported to be closely associated with nodal metastasis.(21) Based on the above findings, the p53 status of tumor stromal fibroblasts may play a very important role in carcinoma progression by IDC.

The purpose of the present study was to determine whether p53 protein expression in tumor stromal fibroblasts is significantly associated with nodal metastasis by IDC, and significantly associated with the outcome of IDC patients with and without adjuvant therapy according to UICC pTNM stage, and triple-negative IDC status. The results indicated that p53 protein expression in tumor stromal fibroblasts is closely associated with the number of nodal metastases and the outcome of IDC patients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cases.  The subjects of the present study were 1042 consecutive patients with IDC of the breast surgically treated at the National Cancer Center Hospital (Tsukiji, Tokyo) between January 2000 and December 2005. The IDC were diagnosed preoperatively by aspiration cytology, mammography, or ultrasonography. Clinical information was obtained from the patients’ medical records after complete histological examination of all IDC. All patients were Japanese women, and they ranged in age from 23 to 77 years (median, 55 years). All had a solitary lesion; 497 patients were premenopausal, and 545 were postmenopausal. Partial mastectomy had been carried out in 462, and modified radical mastectomy in 580. Levels I and II axillary lymph node dissection had been carried out in all patients, and level III axillary lymph node dissection had been carried out in some of the IDC patients.

Of the 1042 patients who did not receive neoadjuvant therapy, 873 had received adjuvant therapy, which consisted of chemotherapy in 209 patients, endocrine therapy in 294 patients, and chemoendocrine therapy in 370 patients. The chemotherapy regimens used were anthracycline-based with or without taxane and non-anthracycline-based, and the endocrine therapy regimens consisted of tamoxifen with or without a gonadotropin-releasing hormone agonist, tamoxifen, with or without an aromatase inhibitor, an aromatase inhibitor alone, or a gonadotropin-releasing hormone agonist alone. There were no cases of inflammatory breast cancer in this series. All tumors were classified according to the pathological UICC-TNM (pTNM) classification.(22) The protocol of this study was reviewed by the institutional review board of the National Cancer Center (20-112), and all patients provided written informed consent.

For pathological examination, the surgically resected specimens were fixed in 10% formalin, and the size and gross appearance of the tumors were recorded. Their size was confirmed by comparison with tumor size on histological slides, and if there was more than one invasive focus, the size of the largest invasive focus was recorded as the invasive tumor size in this study.

Histological examination.  Serial sections of each tumor area were cut from paraffin blocks. One section from each tumor was stained with hematoxylin and eosin (HE) and examined histologically to confirm the diagnosis, and another section was subjected to immunohistochemistry. The following 10 histological factors were evaluated: (1) invasive tumor size (≤20, 20–50, >50 mm); (2) histological grade (1–3);(23) (3) tumor necrosis (absent, present);(24) (4) fibrotic focus (FF) (absent, FF diameter ≤ 8 mm, FF diameter > 8 mm);(7,8) (5) lymphatic invasion (absent, present); (6) blood vessel invasion (absent, present); (8) adipose tissue invasion (absent, length ≤ 2 mm, length > 2 mm);(25) (9) skin invasion (absent, present); and (10) muscle invasion (absent, present).

Immunohistochemistry.  Immunohistochemical staining for estrogen receptors (ER), progesterone receptors (PR), p53, and HER2 products was carried out with an autoimmunostainer (Optimax Plus; BioGenex, San Ramon, CA, USA). Antigen retrieval device for these antibodies and each specimen was immersed in citrate buffer and incubated at 121°C for 10 min. Immunoperoxidase staining was carried out using a labeled streptavidin–biotin (LSAB) staining kit (BioGenex) according to the manufacturer’s instructions. The antibodies used were anti-ER mouse mAb (ER88; BioGenex), an anti-PR mAb (PR88; BioGenex), an anti-HER2 mAb (CB11; BioGnex), and a p53 mAb (DO7; Dako, Glostrup, Denmark). ER88, PR88, and CB11 were already diluted and DO7 was applied at 1:100 dilution. After immunostaining, the sections were counterstained with hematoxylin. Sections of IDC positive for ER, PR, HER2, and p53 were used each time as positive internal or external controls. As a negative control, the primary antibody was replaced with normal mouse immunoglobulin.

Assessment of ER, PR, p53, and HER2 expression.  Slides immunostained for ER, PR, and p53 in tumor cells were scored by the Allred scoring system as described previously.(26–30) Although the validity of the Allred scoring system for assessing expression of ER, PR, and p53 in tumor cells has been demonstrated,(26–30) the number of tumor stromal fibroblasts that express p53 in tumors is relatively small, and the distribution of tumor stromal fibroblasts expressing p53 is scattered even in IDC with tumor stromal fibroblasts having Allred scores of 4–8. We therefore modified the Allred scoring system to assess expression of p53 in tumor stromal fibroblasts by identifying the field with the highest proportion score (PS) and intensity score (IS) for p53 expression in the tumor area (hot spot) by scanning the entire tumor section stained for p53 at medium power (×10 objective and ×10 ocular) (Fig. 1A,B). The highest IS (0, none; 1, weak; 2, intermediate; 3, strong) for expression of p53, not the average IS in the original,(26–30) was assigned for tumor stromal fibroblasts (Figs 1C,D,2A–F), and the highest p53 expression PS (0–5) was then to be evaluated in one high-power field (hot spot, ×40 objective and ×10 ocular) (Figs 1C,D,2G–P). The PS and IS of the tumor stromal fibroblasts were then added to obtain a total score, with possible total score of 0 and ranging from 2 to 8 (Figs 1D,2G–P). When examining tumor stromal fibroblasts for p53 staining, we always confirmed by HE stained specimen whether the cells that show positive staining for p53 is tumor-stromal fibroblasts or not. The HER2 status of the tumor cells was semiquantitatively scored on a 0–3 scale according to the level of HER2 protein expression.(31) Immunohistochemistry was used to score 1025 of the 1042 IDC for ER, PR, or HER2 expression and to score 1026 of them for p53 expression.

image

Figure 1.  Method for immunohistochemical assessment of tumor stromal fibroblasts in invasive ductal carcinoma (IDC). (A–D) First, sections on slides were examined for the presence or absence of p53 expression in tumor stromal fibroblasts in a medium-power field (×10 objective and ×10 ocular or ×20 objective and ×10 ocular), and areas in which tumor stromal fibroblasts showed p53 expression were found. Intensity score (IS) and proportion score (PS) were then assigned for p53 expression in tumor stromal fibroblasts in one high-power field in each medium-power field in which staining was found (×40 objective and ×10 ocular). The high-power field with the highest Allred score (IS + PS) for p53 expression was selected as the hot spot in the tumor. (C) Negative and positive tumor stromal fibroblasts for p53 expression were observed in the same high-power field, the hot spot in this tumor (×40 objective and ×10 ocular). Of the tumor stromal fibroblasts that showed positive nuclear staining for p53, two had a strong IS of 3 for p53, four had an IS of 2, and three had an IS of 1. The IS of tumor stromal fibroblasts for p53 expression in this case was 3. (D) PS for p53 expression. PS ranged from 0 to 5, and the highest PS was recorded as the PS of the case. The IS and PS for p53 expression in tumor stromal fibroblasts were then added to obtain a total score, the Allred score (AS), with total scores of 0 and 2–8. There was a hot spot in the tumor in each of the six IDC cases (a–f). The IDC case with hot spot a had an AS of 0, and the AS of the IDC case with hot spot b was 2. The IDC cases having hot spots c, d, e, and f had AS of 4, 6, 7, and 8, respectively. The IS for p53 in tumor stromal fibroblasts in each case were based on the tumor stromal fibroblasts with the highest IS for p53 expression (arrowheads).

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image

Figure 2.  (A,C,E,G,I,K,M,O) Histological features of tumor stromal fibroblasts, (B,D,F) intensity scores (IS), and (H,J,L,N,P) proportion scores (PS) for p53 expression in tumor stromal fibroblasts of invasive ductal carcinomas (IDC) in high-power fields (×40, hot spots). In general, tumor stromal fibroblasts had spindled acidophilic cytoplasm and oval nuclei, and were mixed with collagen fibers. Nucleoli of tumor stromal fibroblasts were inconspicuous. However, some tumor stromal fibroblasts exhibited epithelioid features, and had enlarged round to oval nuclei containing small nucleoli. Thus, the pathologist should confirm that cells showing p53 expression are tumor stromal fibroblasts or tumor cells not only by immunostaining, but also by hematoxylin–eosin staining. The tumor stroma contained tumor stromal fibroblasts with (A,B) an IS of 1 and (C,D) an IS of 2 for p53 expression (arrowheads). No tumor cells exhibited p53 expression (lower-right corner). (E,F) Tumor stromal fibroblasts with an IS of 3 for p53 expression were observed in the tumor stroma (arrowheads). Tumor cells with an IS of 3 or 2 for p53 expression are also observed (lower-left corner). (G,H) An Allred score (AS) of 3 for p53 expression in tumor stromal fibroblasts. One tumor stromal fibroblast in the high-power field had an IS of 2 for p53 expression (×40, hot spot, arrowhead). Tumor cells with an AS of 8 for p53 were also observed (lower-left corner and upper-right corner). (I,J) Tumor stromal fibroblasts with an IS of 1 or 2 for p53 expression were visible in the tumor stroma (arrowheads), and the PS of the tumor fibroblasts in this case was 2. Thus, the AS of the tumor stromal fibroblasts in the case was 4. Tumor cells were negative for p53 nuclear staining (upper-right corner and lower-left corner). (K,L) Tumor stromal fibroblasts with an IS of 3 or 2 for p53 expression were observed in the tumor stroma (arrowheads). The PS of the tumor stromal fibroblasts for p53 in this case was 3, and the AS of the tumor stromal fibroblasts for p53 expression in this case was 6. Tumor cells with an IS of 3 for p53 expression were also observed (upper-center and lower-right corner). (M,N) Tumor stromal fibroblasts had an IS of 3 or 2 for p53 expression (arrowheads), and the PS of the tumor stromal fibroblasts for p53 was 4. The AS of tumor stromal fibroblasts for p53 expression of this case is 7. No tumor cells are visible. (O and P) Many tumor stromal fibroblasts with an IS of 2 or 1 for p53 expression were observed in the tumor stroma between tumor cell nests (arrows), and their PS for p53 was 5. The AS of the tumor stromal fibroblasts for p53 expression in this case was 7.

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One author (TH) assessed all of the immunohistochemical parameters, and one of three other authors (HT, TS, or YS) identified the immunohistochemical parameters to confirm the IDC immunohistochemical characteristics recorded by TH. Discordant results were reevaluated jointly to reach a consensus. The histological and immunohistochemical examinations were carried out without knowledge of the patients’ outcomes.

Patient outcome and statistical analysis.  Survival was evaluated by follow up for a median period of 52 months (range, 18–102 months) until June 2008. Of the 1042 IDC patients, 924 patients were alive and well, 118 had developed tumor recurrence, and 29 had died of their disease, and an initial distant organ metastasis was observed in 85 of the 118 IDC patients with tumor recurrence. The measurements of tumor recurrence-free survival, initial distant organ metastasis-free survival, and overall survival started on the day of surgery. Tumor relapse was considered to have occurred whenever there was evidence of metastasis.

The Allred scores for ER, PR, and p53 expression in tumor cells and tumor stromal fibroblasts were classified into three categories according to the univariate analyses by the Cox proportional hazard regression model most significantly associated with tumor recurrence: (1) the Allred scores for ER in tumor cells were classified into the three categories 0 or 2, 3–6, and 7 or 8; (2) the Allred scores for PR in tumor cells were classified into the categories 0 or 2, 3–6, and 7 or 8; (3) the Allred scores for p53 in tumor cells were classified into the three categories 0 or 2 or 3, 4–6, and 7 or 8; and (4) the Allred scores for p53 in tumor stromal fibroblasts were classified into the three categories 0 or 2, 3, and 4–8. HER2 expression in tumor cells was classified into the three categories: 0 or 1, 2, and 3.

The 10 histological factors, and the Allred scores for ER, PR, and p53 in tumor cells, Allred scores for p53 in tumor stromal fibroblasts, categories of HER2 expression in tumor cells, and age (≤39 years and >39 years) were analyzed in association with nodal metastases and the outcome of the IDC patients. Univariate analysis associations with the number of nodal metastasess were carried out by ANOVA, and the factors significantly associated with the number of nodal metastases in the univariate analysis were then entered in a multiple regression analysis for multivariate analyses. Univariate analysis associations between the above factors and UICC pathological nodal status (N factor: N0, no nodal metastasis; N1, 1–3 nodal metastases; N2, 4–9 nodal metastases; and N3, 10 or more nodal metastasis) and the outcomes of the IDC patients were carried out using the Cox proportional hazard regression model. The factors significantly associated with outcome in the univariate analyses were then entered together into the multivariate analyses using the Cox proportional hazard regression model. The multivariate analyses were carried out separately in patients with and without adjuvant therapy according to UICC pTNM stage(22) and triple-negative IDC status. The case-wise and step-down method was applied until all of the remaining factors were significant at a P-value below 0.05. As there were fewer than 10 tumor deaths in the group of patients who did not receive adjuvant therapy, the group with UICC pTNM stage I disease, the group with UICC pTNM stage II disease, and the triple-negative-IDC group of patients, it was impossible to carry out multivariate analyses for tumor death in these groups. Survival curves were drawn by the Kaplan–Meier method.(32) All analyses were done with Statistica/Windows software (StatSoft, Tulsa, OK, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Analyses for the number of nodal metastases.  Allred score for p53 in tumor stromal fibroblasts (Fig. 3A), Allred scores for ER, PR, and p53 in tumor cells, invasive tumor size, skin invasion, adipose tissue invasion, histological grade, fibrotic focus, lymph vessel invasion, and blood vessel invasion were significantly associated with the number of nodal metastases in the univariate analyses (data not shown). Invasive tumor size, lymph vessel invasion, skin invasion, p53 Allred score in tumor stromal fibroblasts, blood vessel invasion, histological grade, and Allred score for PR in tumor cells were significantly associated with the number of nodal metastases in the multivariate analyses (Fig. 3B).

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Figure 3.  Associations between (A) the number of nodal metastases and Allred scores for p53 in tumor stromal fibroblasts and (B) factors that were significantly associated with the number of nodal metastases in the multivariate analyses. (A) In invasive ductal carcinoma (IDC) patients, the increase in number of nodal metastases was significantly associated with the Allred scores for p53 in tumor stromal fibroblasts. (B) Multiple regression analysis revealed the factors that were significantly associated with the increase in number of nodal metastases in IDC patients. CI, confidence interval.

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Factors significantly associated with distant organ metastasis and tumor death.  HER2 expression in tumor cells, Allred scores for p53 in tumor cells and in tumor stromal fibroblasts (Fig. 4A), Allred scores for ER in tumor cells, Allred scores for PR in tumor cells, invasive tumor size, histological grade, FF diameter, lymph vessel invasion, blood vessel invasion, UICC pN categories, and UICC pTNM stages were significantly associated with distant organ metastasis and tumor death in the univariate analyses (data not shown). Age was significantly associated with tumor recurrence in the univariate analyses, and skin invasion was significantly associated with tumor death in the univariate analyses (data not shown). Adjuvant therapy, muscle invasion, adipose tissue invasion, and tumor necrosis showed no significant association with tumor recurrence or tumor death in the univariate analyses (data not shown).

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Figure 4.  (A) Distant organ metastasis-free survival curves of invasive ductal carcinoma (IDC) patients according to the Allred scores for p53 in their tumor stromal fibroblasts and (B) the disease-free survival curves of the triple-negative IDC patients. (A) Distant organ metastasis-free survival of IDC patients with Allred scores of 4–8 for p53 in tumor stromal fibroblasts is significantly shorter than that of IDC patients with other Allred scores for p53 in tumor stromal fibroblasts. (B) IDC patients with Allred scores of 4–8 for p53 in tumor stromal fibroblasts had a significantly shorter disease-free survival than IDC patients with Allred scores of 0 or 2 for p53.

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Among the patients who did not receive adjuvant therapy, only histological grade 3 significantly increased the hazard rate (HR) for distant organ metastasis in the multivariate analyses (data not shown).

In the UICC pTNM stage I patients who received adjuvant therapy, Allred scores of 4–8 for p53 in tumor stromal fibroblasts, histological grade 3, and FF diameter > 8 mm significantly increased the HR for distant organ metastasis in the multivariate analyses (Table 1).

Table 1. Multivariate analyses for distant organ metastasis in UICC pTNM stage I invasive ductal carcinoma patients who received adjuvant therapy (= 247)
 CasesDOMR (%)HR95% CIP-value
  1. CI, confidence interval; DOMR, distant organ metastasis rate; HR, hazard rate.

Allred scores for p53 in tumor stromal fibroblasts
 0 or 21482 (1)Referent  
 3380Referent  
 4 to 8619 (15)32.23.4–306.10.003
Histological grade
 1660Referent  
 21232 (2)Referent  
 3629 (15)7.51.6–35.10.011
Fibrotic focus, diameter
 Absent1866 (3)Referent  
 ≤8 mm453 (7)1.70.4–7.60.457
 >8 mm202 (10)9.11.5–56.40.018

In the UICC pTNM stage II patients who received adjuvant therapy, Allred scores of 4–8 for p53 tumor stromal fibroblasts, age ≤39 years, and FF diameter > 8 mm significantly increased the HR for distant organ metastasis in the multivariate analyses (Table 2).

Table 2. Multivariate analyses for distant organ metastasis in UICC pTNM stage II invasive ductal carcinoma patients who received adjuvant therapy (= 435)
 CasesDOMR (%)HR95% CIP-value
  1. CI, confidence interval; DOMR, distant organ metastasis rate; HR, hazard rate.

Allred scores for p53 in tumor stromal fibroblasts
 0 or 22268 (4)Referent  
 3873 (4)1.80.6–4.80.263
 4 to 812218 (15)3.81.8–8.1<0.001
Age (years)
 ≤39417 (17)Referent  
 >3940224 (6)0.30.1–0.70.006
Fibrotic focus, diameter
 Absent26514 (5)Referent  
 ≤8 mm1015 (5)1.50.4–6.40.578
 >8 mm7712 (16)2.81.3–6.00.006

Among the UICC pTNM stage III patients who received adjuvant therapy, p53 Allred scores of 4–8 in tumor stromal fibroblasts significantly increased the HR for distant organ metastasis and tumor death in the multivariate analyses (Table 3). FF diameter > 8 mm, the presence of blood vessel invasion, and category 3 HER2 expression in tumor cells significantly increased the HR for distant organ metastasis, and histological grade 3 significantly increased the HR for tumor death in the multivariate analyses (Table 3).

Table 3. Multivariate analyses for distant organ metastasis, and tumor death in UICC pTNM stage III invasive ductal carcinoma patients who received adjuvant therapy (= 185)
 CasesDOMR (%)HR95% CIP-value
  1. CI, confidence interval; DOMR, distant organ metastasis rate; HR, hazard rate; TDR, tumor death rate; −/−, not significant.

Distant organ metastasis
Allred scores for p53 in tumor stromal fibroblasts
 0 or 2886 (7)Referent  
 3222 (9)0.640.1–4.10.639
 4 to 87425 (34)6.22.7–13.8<0.001
Fibrotic focus, diameter
 Absent9713 (13)Referent  
 ≤8 mm465 (11)0.40.1–1.20.099
 >8 mm4215 (36)3.41.6–7.0<0.001
Blood vessel invasion
 Absent14519 (13)Referent  
 Present3814 (37)2.71.3–5.70.006
HER2 expression
 0 or 111919 (16)Referent  
 2384 (11)0.70.2–2.30.588
 32710 (37)2.41.1–5.30.023
Tumor death
Allred scores for p53 in tumor-stromal fibroblasts
 0 or 2881 (1)Referent  
 3220Referent  
 4 to 87416 (22)18.12.4–139.50.005
Histological grade
 1300Referent  
 2763Referent  
 37914 (18)3.81.1–12.90.038

Among the triple-negative IDC patients, Allred scores of 4–8 for p53 in tumor stromal fibroblasts (Fig. 4B), UICC pN2 and pN3, and age ≤ 39 years significantly increased the HR for tumor recurrence in the multivariate analysis (Table 4).

Table 4. Clinicopathological factors significantly associated with tumor recurrence in triple-negative invasive ductal carcinoma patients in multivariate analyses (= 74)
 CasesTRR (%)HR95% CIP-value
  1. CI, confidence interval; HR, hazard rate; TRR, tumor recurrence rate; UICC pN0, no nodal metastasis; UICC pN1, one to three nodal metastases; UICC pN2, four to nine nodal metastases; UICC pN3, 10 or more nodal metastases. Triple-negative residual invasive ductal carcinoma means: (1) Allred scores for estrogen receptor of 0 or 2; (2) Allred score for progesterone receptor of 0 or 2; and (3) HER2 expression category of 0 or 1.

Allred scores for p53 in tumor-stromal fibroblasts
 0 or 2302 (7)Referent  
 370Referent  
 4 to 83713 (35)12.02.3–62.00.003
UICC pN category
 pN0413 (7)Referent  
 pN1174 (24)1.40.3–6.90.701
 pN294 (44)7.82.0–30.60.004
 pN374 (57)26.86.0–122.6<0.001
Age (years)
 ≤3963 (50)Referent  
 >396812 (18)0.20.04–0.80.022

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patocs et al.(21) showed a significant association between p53 mutations in tumor stroma and nodal metastasis in patients with sporadic breast cancer, and the results of the multivariate analyses in the present study clearly confirmed that p53 expression in tumor stromal fibroblasts, but not in tumor cells, is an important independent factor associated with the number of nodal metastases by IDC.

The present study also clearly demonstrated that Allred scores of 4–8 for p53 expression in tumor stromal fibroblasts significantly increased the HR for distant organ metastasis and tumor death in the IDC groups independent of UICC pTNM stage or triple negativity. Thus, Allred scores of 4–8 for p53 in tumor stromal fibroblasts can be concluded to be a very important factor for accurately predicting the outcome of IDC patients independent of UICC pTNM stage or triple negativity, and it can be concluded that the modified method that we used to assign Allred scores to p53 expression in tumor stromal fibroblasts should be used to accurately evaluate the malignant potential of IDC from the standpoint of the tumor stroma.

In the present study we did not test Allred scores for p53 expression for associations with the presence of p53 gene abnormalities in the tumor stromal fibroblasts. Although p53 mutations in tumor stromal fibroblasts are relatively common in primary breast cancer and other cancers and have a positive effect on cancer growth,(18–21,33) some studies show no p53 mutations observed in the tumor stroma of breast cancer,(34,35) and the possibility of technical problems (e.g. PCR artifacts for p53 gene abnormalities) is raised by Campbell et al.(36) Thus, although the mechanism responsible for increasing the malignant potential of IDC related to the expression of p53 in tumor stromal fibroblasts should be investigated from the standpoint of p53 gene abnormalities, p53 immunoreactivity in tumor stromal fibroblasts may in fact reflect specific reactive changes within the stroma that are related to the outcome of patients with IDC.

The results of the present study clearly showed that FF diameter, age, blood vessel invasion, and UICC pN2 were good prognostic factors. In previous studies we found that FF diameter was a significant prognostic factor for IDC patients,(7–10,13,37) and the significant prognostic power of FF diameter in the IDC group was confirmed in the present study. Thus, one can conclude that FF diameter is an important histological outcome predictor for IDC patients, and biological characteristics of tumor stroma, e.g. tumor stromal fibroblasts expressing p53, FF, play a very important role in tumor progression of IDC of the breast.

In conclusion, this is the first study to clearly demonstrate that p53 expression by tumor stromal fibroblasts is strongly associated with the number of nodal metastases and the outcome of IDC patients. The modified Allred scoring system is very suitable for accurately assessing p53-expressing tumor stromal fibroblasts in IDC independent of adjuvant therapy, UICC pTNM stage, or the HR or HER2 status of the IDC. The p53 expression in tumor stromal fibroblasts will probably be a very important target for tumor gene therapy of IDC, although it will be necessary to confirm that the p53 Allred score also provides significant prognostic power for IDC patients in a prospective study.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) (C) (19590378, 21590393) and was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare of Japan (20-16).

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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