Int. J. Cancer (Pred. Oncol.): 89, 39 – 43 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer CORRELATION OF CYCLIN D1 MRNA LEVELS WITH CLINICO-PATHOLOGICAL PARAMETERS AND CLINICAL OUTCOME IN HUMAN BREAST CARCINOMAS Toshiaki UTSUMI1*, Noriko YOSHIMURA2, Morito MARUTA1, Shinji TAKEUCHI3, Jiro ANDO4, Yoshikazu MIZOGUCHI5 and Nobuhiro HARADA2 1 Department of Surgery, Fujita Health University School of Medicine, Toyoake, Japan 2 Department of Biochemistry, Fujita Health University School of Medicine, Toyoake, Japan 3 Marumo Hospital, Nagoya, Japan 4 Department of Surgery, Tochigi Cancer Center, Utsunomiya, Japan 5 Department of Pathology, Fujita Health University School of Medicine, Toyoake, Japan In order to evaluate the prognostic significance of cyclin D1 mRNA expression in mammary neoplasia, its levels were measured in 97 breast cancers by reverse transcription-polymerase chain reaction (PCR) using fluorescent primer and standard RNA along with estrogen receptor (ER). The median value of cyclin D1 mRNA was 1.60 amol/g RNA (range, 0.01 to 5.63 amol/g RNA). ER mRNA was detectable in 70 breast cancer samples (72.2%) and cyclin D1 mRNA levels were significantly higher in ER mRNA-positive than in ER mRNA-negative tumors (p ⴝ 0.009). Furthermore, cyclin D1 mRNA levels were significantly (p ⴝ 0.001) lower in patients who experienced a recurrence during the follow-up period (mean of 40.8 months, median of 39 months) compared with those with no evidence of recurrent disease (mean of 49.2 months, median of 48 months), and in those who died from disease (mean follow-up period of 30.5 months, median of 26 months) than in the survivors (mean of 50.5 months and median of 48 months) (p ⴝ 0.005). Setting the median value (ⴝ1.60 amol/g RNA) as the cutoff point, expression was significantly associated with relapse-free survival (p ⴝ 0.002). Similarly, a significant correlation was observed between the cyclin D1 mRNA level and overall survival (p ⴝ 0.015). The expression was found to be an independent factor for predicting relapse-free survival using multivariate analysis. Int. J. Cancer (Pred. Oncol.) 89:39 – 43, 2000. © 2000 Wiley-Liss, Inc. D-type cyclins are strongly implicated in controlling progression through the G1 phase of the cell cycle. Three closely related human D-type cyclins activate cyclin-dependent kinases (Cdks), Cdk4 and Cdk6, although they have specialized functions in distinct cell types (reviewed by Sherr, 1993). As a G1 cyclin, cyclin D1 was identified originally as a putative proto-oncogene, BCL1/ PRAD1, located on chromosome 11q13, as a suppressor of yeast G1 cyclin mutations and as a delayed early response gene induced by colony-stimulating factor 1 (Sherr, 1993). Remarkable overexpression of cyclin D1 has been observed in human neoplasms (reviewed by Donnellan and Chetty, 1998). It is likely to promote cell proliferation and differentiation by shortening the G1-S transition (reviewed by Sherr, 1994). A further hint that cyclin D1 could be associated with tumorigenesis came from the fact that it is inducible by activated myc oncogene (Daksis et al., 1994). Cyclin D1 may also be a mediator of apoptotic neuronal cell death (Kranenburg et al., 1996), and Pagano et al. (1994) have shown that a transient overexpression in fibroblasts arrests the cells in the G1-phase of the cell cycle. In senescent fibroblasts, an increased level of cyclin D1 has been found (Dulic et al., 1993). These reports indicate 2 opposing impacts of cyclin D1 overexpression in cell cycle control. Breast cancer is a worldwide problem which urgently requires solutions; in many countries it is still the most common cause of malignancy associated death. Early stage detection and treatment has become possible, but this has meant difficulty in obtaining adequate tumor tissues for biological studies of estrogen receptor (ER) status and also measurement of other prognostic parameters. However, the development of molecular biology techniques has allowed various biochemical factors to be assayed in small tissue specimens, and we have reported the biological significance of enzymes concerned with estrogen synthesis (Utsumi et al., 1996, 1999). In the present study, to investigate the role of cyclin D1 in breast cancers, we assessed the mRNA levels of cyclin D1 and ER using reverse transcription-polymerase chain reaction (RT-PCR) analysis in 97 cases. Studies in mice and humans have linked cyclin D1 with steroid-induced proliferation of mammary epithelial cells (Donnellan and Chetty, 1998). Our present results indicate that cyclin D1 expression is associated with ER mRNA levels, prognostic factors, and patient outcome. MATERIAL AND METHODS Patients and samples Material for this study was obtained from 97 patients with primary breast carcinomas who underwent curative surgery at Fujita Health University Hospital, Marumo Hospital, and Tochigi Cancer Center between 1990 and 1994. The average age of the patients was 52.2 ⫾ 9.5 years (mean ⫾ standard deviation), with a range of 33 to 77 years. Fifty patients received adjuvant chemotherapy, and 65 were given adjuvant endocrine therapy. Disease recurrence was documented on the basis of physical examination, radiological and laboratory tests and/or other relevant diagnostic procedures. The median follow-up period for all patients was 46 months, with a range of 7 to 95 months. The tumor types of the 97 patients were classified by pathologists according to the World Health Organization scheme for typing breast tumors. Histologically, there was 1 case each of ductal carcinoma in situ, mucinous carcinoma, and invasive lobular carcinoma and 94 of invasive ductal carcinomas. Fifty-two cases were node negative and 45 were node positive. Each tumor, exclusive of 1 ductal carcinoma in situ and 1 invasive lobular carcinoma, was graded in parallel according to the criteria of Bloom and Richardson (1957). Tumor size was measured at surgery by the operating physicians. Immediately following surgical removal, the specimens were frozen in liquid nitrogen and then stored at ⫺80°C until use. ERs were assayed by means of the dextran-coated charcoal method with a cutoff value of 5 fmol/mg protein. This research project was approved by the Medical Ethics Committee of Fujita Health University School of Medicine. Preparation of total RNA Frozen tissues were homogenized in 5 M guanidine thiocyanate containing 5 mM sodium citrate and 0.5% sodium sarcosyl, and total RNA fractions were prepared from the homogenates, as described previously (Utsumi et al., 1996, 1999). The RNA con*Correspondence to: Department of Surgery, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. Fax: ⫹81-562-938311. E-mail: [email protected] Received 7 June 1999; Revised 23 July 1999 40 UTSUMI ET AL. centration was determined from the spectrophotometric absorption at 260 nm. Quantitation of cyclin D1 mRNA Quantitative analysis of cyclin D1 mRNA in the RNA fractions was carried out by RT-PCR using a fluorescent primer as described previously (Utsumi et al., 1996, 1999). In brief, oligonucleotides of antisense primer, H-D1-1R (5⬘-GTCACACTTGATCACTCTGG-3⬘) for reverse transcription, and antisense (5⬘-CCAGGTTCCACTTGAGCTTG-3⬘) and sense (5⬘-CCTACTTCAAATGTGTGCAG-3⬘) primers, H-D1-2R and H-D1-3F for PCR, respectively, were synthesized. The sense primer H-D1-3F for PCR was labeled with a fluorescent dye, FAM (Perkin-Elmer, Norwalk, CT), after connection with Aminolink 2 (Perkin-Elmer). The coding sequence between the 2 PCR primer sites is located 5⬘ upstream of the reverse transcription primer site in the cyclin D1 transcript, and is interrupted by an intron in the gene. Standard cyclin D1 RNA was synthesized in vitro with T7 RNA polymerase using cyclin D1 cDNA as a template, purified on an anion exchange column of Qiagen (Chatsworth, CA), and then quantitated from the absorbance at 260 nm. Total RNA (1–2 g) and standard cyclin D1 RNA (0.5 amol) were subjected to reverse transcription with 5 units of RAV-2 reverse transcriptase (Takara Shuzo, Kyoto, Japan) and the specific antisense primer H-D1-1R at 42°C for 40 min. The resulting cDNAs were amplified by PCR using the fluorescent dye-labeled primer H-D1-3F and H-D1-2R. The PCR conditions were: denaturation at 94°C for 20 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec for 19 cycles. Fluorescent PCR products were analyzed on 2% agarose gels with a Gene Scanner 362 Fluorescent Fragment Analyzer (PerkinElmer). The amount of cyclin D1 mRNA in each tissue RNA was calculated by comparing the peak area of the fluorescent products with that of standard cyclin D1 RNA. Similarly, standard ER RNA was synthesized in vitro using ER cDNA as a template, and quantitative analysis of ER mRNA was also carried out by RT-PCR, in which the antisense primer (5’GCCTTTGTTACTCATGTGCC-3⬘) was employed for reverse transcription, and antisense (5⬘-GTGTCTGTGATCTTGTCCAG3⬘) and fluorescent dye-labeled sense (5⬘-CTGATGATTGGTCTCGTCTG-3’) primers for PCR. RT-PCR analyses were performed under the condition that the fluorescent peak areas of PCR products corresponding to cyclin D1 and ER mRNAs were proportional to the amounts of total RNAs added as to templates. This proportionality between their amounts and their fluolescent peak areas was observed over a wide range of 0.002–10 amol and 0.002–10 amol for cyclin D1 and ER mRNAs, respectively, in these assays. Statistics Statistical analyses were carried out with SAS-REL.6.12. software. The Spearman’s correlation coefficient was used to investigate correlations among different clinico-pathological variables. Mean levels of cyclin D1 mRNA were compared using the MannWhitney U test. Relapse-free and overall survival curves were generated using the method of Kaplan and Meier. Survival comparisons were made with the log-rank test, the Wilcoxon test, the proportional hazards regression model, and proportional hazards (Cox) multiple regression. The event considered in our analysis of relapse-free survival was first recurrence of disease. Overall survival refers to survival with or without recurrence of disease. Relapse-free survival and overall survival were calculated from the date of first surgery to the date of clinical or pathological relapse or death. The cutoff for significance was taken as p ⫽ 0.05. RESULTS Cyclin D1 mRNA in breast cancer tissues Cyclin D1 mRNA levels in the breast tissues from 97 patients with breast cancer were determined by quantitative RT-PCR analysis. Figure 1 shows the distribution of the cyclin D1 mRNA levels FIGURE 1 – Distribution of cyclin D1 mRNA levels in the 97 primary breast cancer tumors. The class interval is 1 amol/g RNA. The mean of 2.13 amol/g RNA is indicated by a solid arrow, and the median (1.60 amol/g RNA) is indicated by an open arrow. in the 97 breast tissues. The distribution was not normal, and there was a median value of 1.60 amol/g RNA (range, 0.01–5.63 amol/g RNA). The mean value was 2.13 ⫾ 1.64 amol/g RNA (mean ⫾ standard deviation). ER mRNA expression and ER protein status In our series, 57 cases were ER protein positive, 37 were ER negative, and 3 were unknown. The ER mRNA levels in the breast tissues from 97 patients with breast cancer were also determined by RT-PCR analysis. The ER calculated from the peak area of the fluorescent PCR product by comparison with that of a standard ER RNA (0.5 amol). The distribution of ER mRNA was not normal (data not presented), and ER mRNA were detectable in 70 samples (72.2%) and 27 (27.8%) were negative. A positive correlation was observed between the ER mRNA expression and the ER protein status assessed by the dextran-coated charcoal method (p ⫽ 0.0001, r ⫽ 0.670, n ⫽ 94). Correlation of cyclin D1 mRNA expression with clinico-pathological features Table I shows the clinical profiles of the 97 patients, comparing cases at or above the median cyclin D1 mRNA level with those below the median. Cyclin D1 mRNA expression was not found to be significantly correlated with any clinical parameters except ER mRNA expression and endocrine therapy history. There was no significant association between cyclin D1 mRNA and age (p ⫽ 0.240, r ⫽ ⫺0.121, n ⫽ 97), menopausal status (p ⫽ 0.086, r ⫽ 0.175, n ⫽ 97), tumor size (p ⫽ 0.485, r ⫽ 0.072, n ⫽ 97), nodal status (p ⫽ 0.361, r ⫽ 0.094, n ⫽ 97), chemotherapy history (p ⫽ 0.131, r ⫽ 0.154, n ⫽ 97) or histological grade (p ⫽ 0.080, r ⫽ 0.181, n ⫽ 95). There was a significant but only weak association between cyclin D1 mRNA and ER mRNA status (p ⫽ 0.004, r ⫽ 0.293, n ⫽ 97) and endocrine therapy history (p ⬍ 0.001, r ⫽ 0.344, n ⫽ 97). The relation between cyclin D1 and endocrine therapy history may not be relevant, since endocrine therapy is usually given to patients on the basis of their ER status which was associated with the cyclin D1 status. The levels of cyclin D1 mRNA in ER mRNA-positive tumors were significantly (p ⫽ 0.009) higher than those in ER mRNA-negative tumors (Table II). There were also significantly (p ⬍ 0.001) higher cyclin D1 mRNA levels in ER protein positive breast cancers compared with ER protein negative tumors (data not shown). A total of 20 patients had recurrent disease, and 15 had died by the time of the analyses. Cyclin D1 mRNA on the basis of the median and the mean was then examined in association with the disease recurrence and death during the follow-up period (Table III). There were significantly (p ⫽ 0.001) lower cyclin D1 mRNA levels in patients who experienced a recurrence during the fol- CYCLIN D1 EXPRESSION IN HUMAN BREAST CANCER 41 TABLE I – CLINICAL PROFILES OF THE 97 BREAST CANCER PATIENTS Cyclin D1 mRNA Number of patients Mean age (years) ER mRNA Negative Positive Tumor size ⱕ2.0 cm ⱖ2.1 cm Nodal status Negative Positive Histological grade 1 2 3 Adjuvant chemotherapy No Yes Adjuvant endocrine therapy No Yes Number of recurrences Number of deaths during follow-up Number of survivors during follow-up Mean follow-up time for deceased patients (months) Mean follow-up time for survivors (months) ⱕ1.6 amol/g RNA ⬎1.6 amol/g RNA 49 51.0 ⫾ 9.0 48 53.4 ⫾ 9.9 20 (40.8%) 29 (59.2%) 7 (14.6%) 41 (85.4%) 17 (33.3%) 32 (66.7%) 20 (41.8%) 28 (58.2%) 24 (49.0%) 25 (51.0%) 28 (56.4%) 20 (43.6%) 5 (10.4%) 28 (58.3%) 15 (31.3%) 7 (14.9%) 34 (72.3%) 6 (12.8%) 20 (40.8%) 29 (59.2%) 27 (56.3%) 21 (43.7%) 24 (49.0%) 25 (51.0%) 17 13 8 (16.7%) 40 (83.3%) 3 2 36 46 32.0 ⫾ 20.6 21.0 58.2 ⫾ 17.3 44.5 ⫾ 11.0 TABLE II – CYCLIN D1 MRNA VALUES IN BREAST CANCER PATIENTS WITH RESPECT TO ER MRNA EXPRESSION ER mRNA Number Negative Positive 27 70 Cyclin D1 mRNA (amol/g RNA) Mean ⫾ SD Median 1.42 ⫾ 1.33 2.41 ⫾ 1.68 0.97 1.91 p value 0.009 TABLE III – CYCLIN D1 MRNA VALUES IN BREAST CANCER PATIENTS WITH RESPECT TO CLINICAL OUTCOME WITHIN THE FOLLOW-UP PERIOD Number Recurrence No Yes Deceased No Yes Cyclin D1 mRNA (amol/g RNA) p value Mean ⫾ SD Median 77 20 2.40 ⫾ 1.64 1.12 ⫾ 1.24 1.88 0.75 0.001 82 15 2.32 ⫾ 1.64 1.12 ⫾ 1.33 1.88 0.57 0.005 low-up period (mean of 40.8 months and median of 39 months) compared with those with no evidence of disease (mean of 49.2 months, median of 48 months). We further found that the subset, who died after recurrence during the follow-up period (mean of 30.5 months, median of 26 months), had significantly (p ⫽ 0.005) lower cyclin D1 mRNA compared with the survivors (mean of 50.5 months, median of 48 months). Prognostic analysis For statistical evaluation, the patients were divided into 2 groups on the basis of the level of cyclin D1 mRNA expression. Most prognostic factors are usually considered as dichotomized, discontinuous variables. Therefore, to evaluate the cyclin D1 mRNA level as a prognostic factor of relapse-free survival in breast cancer, the median value was used as a cutoff to define “low” and “high” expression. Figure 2 shows Kaplan-Meier survival curves FIGURE 2 – Relapse-free and overall survival of 97 patients with breast cancer according to their cyclin D1 mRNA levels. The cutoff value for low cyclin D1 mRNA was the median. There were 48 patients in the high group, and 49 patients in the low group. Both curves were generated using the method of Kaplan-Meier. for relapse-free survival dichotomized by cyclin D1 mRNA levels. Patients with high levels of cyclin D1 mRNA showed significantly better relapse-free survival compared with those with low levels of the mRNA. The intergroup relationships were significantly different by both the log-rank (p ⫽ 0.002) and the Wilcoxon tests (p ⫽ 0.003). When a more optimal cutoff value (⫽1.30 amol/g RNA) derived by the minimum p-value approach (Altman et al., 1994) was used, the difference between the 2 groups was more distinct (p ⬍ 0.001, log-rank test). Overall survival was also positively correlated with the tissue level of cyclin D1 mRNA and reached significance when the median level of cyclin D1 mRNA was used as the cutoff value (p ⫽ 0.015, log-rank test and p ⫽ 0.022, Wilcoxon test). When cyclin D1 mRNA groups were divided into tertiles [bottom tertile, ⱕ0.95 amol/g RNA (n ⫽ 32); middle tertile, ⱕ2.90 amol/g RNA (n ⫽ 32); upper tertile, ⬎2.90 amol/g RNA (n ⫽ 33)], intergroup differences in relapse-free survival was also distinct (p ⫽ 0.008, log-rank test and p ⫽ 0.013, Wilcoxon test; Fig. 3). Overall survival was also positively correlated with the tissue levels of cyclin D1 mRNA and reached significance when the groups were divided into tertiles (p ⫽ 0.040, log-rank test and p ⫽ 0.060, Wilcoxon test). On univariate analysis, several variables, including the cyclin D1 mRNA level, showed significant correlation with prognosis. Nodal status, cyclin D1 mRNA, and histological grade were found to be correlated with relapse-free survival (p ⫽ 0.003, p ⫽ 0.006 and p ⫽ 0.033, respectively; Table IV). There was also a significant correlation between overall survival and nodal status, cyclin 42 UTSUMI ET AL. D1 mRNA, and histological grade (p ⫽ 0.005, p ⫽ 0.029 and p ⫽ 0.019, respectively) in this population of patients. All variables were taken into account through a stepwise analysis. Nodal status emerged as a strong independent predictor of relapse-free survival [p ⫽ 0.008, relative risk ⫽ 4.410, 95% confidence interval (CI) ⫽ 1.471–13.221], and cyclin D1 mRNA level came next (p ⫽ 0.012, relative risk ⫽ 4.919, 95% CI ⫽ 1.411– 17.142), whereas histological grade was not significant (Table IV). On multivariate analysis of overall survival, the model gave nodal status (p ⫽ 0.002, relative risk ⫽ 13.354, 95% CI ⫽ 2.696 – 66.136) and ER mRNA status (p ⫽ 0.025, relative risk ⫽ 3.823, 95% CI ⫽ 1.187–12.317) as independent prognostic factors, whereas histological grade and cyclin D1 mRNA status were not significant. DISCUSSION In many types of human tumor cells, overexpression of cyclin D1 or deregulation of cyclin D1 contributes to oncogenic transformation in vitro and in vivo (Sherr, 1993; Donnellan and Chetty, 1998). To examine to what extent expression of cyclin D1 might be relevant to clinical outcome, in cases of breast cancers the present quantitative RT-PCR analysis was performed. Although expression varied widely among individuals, statistical analysis provided evidence that the level of cyclin D1 transcripts in human breast cancers may be a useful prognostic indicator. In the present study, using molecular biology techniques, we showed a good correlation between cyclin D1 mRNA expression and relapse-free or overall survival. However, we were not able to show any significant correlation with age, histological grade, tumor size, or node metastasis. There is at present much controversy surrounding the potential role of cyclin D1 in human neoplasia. In laryngeal and head and neck carcinomas, its overexpression has been shown to be associated with advanced local invasion and presence of lymph node metastasis (Donnellan and Chetty, 1998). In breast cancer, previous immunohistochemical studies indicated a positive correlation between cyclin D1-positivity and good clinical outcome (Gillett et al., 1996; Pelosio et al., 1996). This was, however, not confirmed by other investigators (Michalides et al., 1996; van Diest et al., 1997). The mechanism of action of cyclin D1 is not fully understood and is now being intensively studied. Nevertheless, it is clear that the D-type cyclins are critical modulators of the G1 phase of the cell cycle and transition through the G1/S restriction point (Sherr, 1994). Mitogenic growth factors such as epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor-I promote progression of the cell cycle by enhancing cyclin D-Cdk4 and cyclin D-Cdk6 complex formation and kinase activities during the G1 phase (Sherr, 1994; Grana and Reddy, 1995). Cyclin D proteins, most notably cyclin D1, are thus at a key step in determining whether a cell commits to mitogenesis, and several observations suggest that it also has an important role in promoting the growth of certain human malignancies and in maintaining the transformed phenotype (Donnellan and Chetty, 1998). How can the present results be explained in view of the outcome of our prognostic study? The reasons for the paradoxical behavior of cyclin D1 are unclear but under certain circumstances, cyclin D1 can act as a negative rather than a positive factor. Indeed, it has been argued that an excess of cyclin D1 may be toxic to cells (Quelle et al., 1993). Overexpression may indeed reduce viability. In the present study, we found that cyclin D1 mRNA levels were significantly higher in ER mRNA-positive tumors than in negative ones, in agreement with previous immunohistochemical findings (Michalides et al., 1996; van Diest et al., 1997). The mechanisms FIGURE 3 – Relapse-free and overall survival of 97 the patients with breast cancer by tertiles of cyclin D1 mRNA (see text). There were 32 patients in the lower tertile, 32 patients in the middle tertile, and 33 patients in the upper tertile. TABLE IV – MULTIVARIATE ANALYSIS OF DESCRIPTORS WITH RESPECT TO RELAPSE-FREE AND OVERALL SURVIVAL Relapse-free survival Multivariate1 Variable Univariate p value p value Cyclin D1 mRNA (low/high) Nodal status (positive/negative) ER mRNA (negative/positive) Tumor size (ⱖ2.1 cm/ⱕ2.0 cm) Histological grade3 (3/1 and 2) Menopausal status (pre-/post-) Age (⬍50 years/ⱖ50 years) 0.006 0.003 0.400 0.093 0.033 0.338 0.740 0.012 0.008 ⬎0.2 0.174 0.144 ⬎0.2 ⬎0.2 Overall survival Multivariate1 Relative risk Univariate p value p value Relative risk 4.919 (1.411–17.142)2 4.410 (1.471–13.221)2 — — — — — 0.029 0.005 0.058 0.202 0.019 0.172 0.389 0.106 0.002 0.025 0.137 0.122 ⬎0.2 ⬎0.2 — 13.354 (2.696–66.136)2 3.823 (1.187–12.317)2 — — — — 1 Ninety-five patients were analyzed since in 2 cases information of histological grade was lacking.–2Values in parentheses are 95 percent confidence intervals.–3A case of ductal carcinoma in situ and a case of invasive lobular carcinoma were excluded from this analysis. CYCLIN D1 EXPRESSION IN HUMAN BREAST CANCER underlying increase of cyclin D1 expression in human breast cancer remain unknown at present. The cyclin D1 gene is known to be often amplified or over-expressed in human breast carcinomas. Barbareschi et al. (1997) showed that gene amplification of cyclin D1 is related to high immunocytochemical expression of cyclin D1 and pRB, and that high cyclin D1 expression is associated with positive ER immunoreactivity, suggesting that overexpression of cyclin D1 observed in breast carcinomas may be more frequently due to ER-related up-regulation than to gene amplification. Treatment of breast cancer cells with anti-estrogens inhibits pRB phosphorylation (Watts et al., 1995), while estrogen induces significant phosphorylation of this key component of cyclin-Cdk complexes (Altucci et al., 1996; Prall et al., 1997). This indicates 43 that cyclin-Cdk complexes are likely to be targets of estrogen action. 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