Gou Watanabe

The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library

Tumor suppressor p53 forms a homo-tetramer through its COOH-terminal oligomerization domain and acts as a sequence-specific transcription factor. We have analysed the interrelation among the transcriptional activities, the structure and the cancer-related mutations in the oligomerization domain by using a comprehensive missense mutation library. Here, we examined the ability of 184 mutant p53s in the domain to form an oligomer by expressing these mutant p53s in yeast, and compared the data with the previous information. We showed that specific residues in the α-helix and the β-strand of the oligomerization domain were critical for both oligomer formation and sequence-specific transactivation, and the activities were closely related. In particular, the α-helix was more sensitive to amino-acid substitutions than the β-strand. We found identity in the interrelation between the two activities, that is, monomer mutants were transcriptionally inactive whereas dimer and tetramer mutants retained their transcriptional activities. In TP53 mutation databases, a small number of mutations have been reported in this domain. Surprisingly, most do not encode p53s defective in functional properties. These results indicate that, although oligomer formation is essential for p53 transactivation function, the inactivation of oligomer formation and therefore the inactivation of transactivation may not be essential for tumor suppression by p53 because they do not lead to oncogenic proteins.

Prognostic significance of tumor-infiltrating CD8 + and FOXP3 + lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

IntroductionThe status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).MethodsOne hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.ResultsTNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).ConclusionsThis is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Luminal-type breast cancer: correlation of apparent diffusion coefficients with the Ki-67 labeling index.

PURPOSE To evaluate the correlation between apparent diffusion coefficient ( ADC apparent diffusion coefficient ) values and the Ki-67 labeling index for luminal-type (estrogen receptor-positive) breast cancer not otherwise specified ( NOS not otherwise specified ) diagnosed by means of biopsy. MATERIALS AND METHODS The institutional review board approved this retrospective study, and the requirement for informed consent was waived. Between December 2009 and December 2012, 86 patients with 86 lesions with luminal-type invasive breast cancer NOS not otherwise specified underwent magnetic resonance imaging, including dynamic contrast material-enhanced imaging and diffusion-weighted imaging with b values of 0 and 1000 sec/mm(2). Conventional measurement of the minimum and mean ADC apparent diffusion coefficient s by placing regions of interest and histogram analysis of pixel-based ADC apparent diffusion coefficient data of the entire tumor were performed by two observers independently and correlated with the Ki-67 labeling index of surgical specimens. RESULTS For the interobserver reliability, interclass correlation coefficients for all parameters with the exception of the minimum ADC apparent diffusion coefficient exceeded 0.8, indicating almost perfect agreement. The minimum ADC apparent diffusion coefficient and mean ADC apparent diffusion coefficient and the 25th, 50th, and 75th percentiles of the histograms showed negative correlations with the Ki-67 labeling index (r = -0.49, -0.55, -0.54, -0.53, and -0.48, respectively). Receiver operating characteristic curve analysis for the differential diagnosis between the high-proliferation (Ki-67 ≥ 14; n = 44) and low-proliferation (Ki-67 < 14; n = 42) groups revealed that the most effective threshold for the mean ADC apparent diffusion coefficient was lower than 1097 × 10(-6) mm(2)/sec, with sensitivity and specificity of 82% and 71%, respectively. The area under the receiver operating characteristic curve (AUC) was 0.81 for the mean ADC apparent diffusion coefficient . There were no significant differences in the AUC among the parameters. CONCLUSION Considering convenience for routine practice, the authors suggest that the mean ADC apparent diffusion coefficient of the conventional method would be practical to use for estimating the Ki-67 labeling index.

αB‐crystallin: A novel p53‐target gene required for p53‐dependent apoptosis

The p53 protein is a transcription factor that trans‐activates various genes in response to DNA‐damaging stress. To search for new p53‐target genes, we applied a cDNA microarray system using two independent p53‐inducible cell lines, followed by in silico analysis to detect p53 response elements. Here, we report on crystallin alpha B gene (CRYAB), which encodes αB‐crystallin, and is one of the genes directly trans‐activated by p53. We confirmed it is directly transcribed by p53 using promoter analysis, deletion reporter assay, ChIP assay and EMSA. αB‐crystallin is also upregulated in a p53‐dependent manner and binds to the DNA‐binding domain of p53. Overexpression of αB‐crystallin increased p53 protein and, in contrast, repression of αB‐crystallin decreased p53 protein. Interestingly, both overexpression and repression of αB‐crystallin reduced p53‐dependent apoptosis. In conclusion, we identified that αB‐crystallin was a novel p53‐target gene and required for p53‐dependent apoptosis using two independent p53‐inducible cell lines. This is the first report associating p53 directly with a heat shock protein through trans‐activation and physical interaction. (Cancer Sci 2009; 100: 2368–2375)

Combined Immunohistochemistry of PLK1, p21, and p53 for Predicting TP53 Status: An Independent Prognostic Factor of Breast Cancer.

It is difficult to predict the TP53 status by p53 immunohistochemistry (IHC). We aimed to improve the accuracy of p53 IHC with p53-regulated proteins for predicting the TP53 mutation status. TP53 mutations were detected in 19 of 38 breast cancer patients (50%). Five of 7 cases of protein-truncating mutation of TP53 were completely negative for p53 IHC, whereas 11 of 12 cases of TP53 point mutation were strongly positive for p53 IHC. Therefore, to avoid false negatives, we extracted p53-dependent universally downregulated genes using microarray analysis from 38 breast cancer patients and 2 p53-inducible cell lines. From 9 commonly repressed genes, we evaluated 3 genes, baculoviral IAP repeat-containing 5 (BIRC5), polo-like kinase 1 (PLK1), and BUB1 mitotic checkpoint serine/threonine kinase (BUB1), which were previously identified as p53-dependent repressed genes. PLK1≥Allred total score (TS) 5 showed the highest correlation with TP53 mutation. To decrease false positivity, we evaluated p21 IHC. Although strong staining of p21 was observed in 4 cases (10.5%), all 4 were wild-type TP53. Thus, p53 mutation-like (p53mt-like) IHC was identified by p53 TS7,8 with PLK1≥TS 5 and p21 TS⩽6. p53 mt-like IHC correlated with TP53 mutation (predictive value=0.94). In other 157 breast cancer cases, p53 mt-like was an independent prognostic marker in multivariate analysis and a strong prognostic factor. Stratification with p53 mt-like IHC identified patients with a poorer prognosis. In conclusion, we identified reliable IHC conditions to predict the TP53 status of breast cancer patients.

Contribution of autophagic cell death to p53-dependent cell death in human glioblastoma cell line SF126

Apoptosis and autophagic cell death are programmed cell deaths that are involved in cell survival, growth, development and carcinogenesis. p53, the most extensively studied tumor suppressor, regulates apoptosis and autophagy by transactivating its downstream genes. It also stimulates the mitochondrial apoptotic pathway and inhibits autophagy in a transactivation‐independent manner. However, the contribution of apoptosis and autophagic cell death to p53‐dependent cell death is unclear. Using wild‐type (WT) and mutant (MT) p53 inducible cell lines in TP53‐null SF126 glioblastoma cells, we examined the apoptosis and autophagic cell death induced by p53. WT p53 expression in SF126 cells induced apoptosis and autophagy, and reduced the cell number. An autophagy inhibitor reduced autophagy, increased the S‐phase fraction, and attenuated the inhibition of cell proliferation induced by WT p53. Pan‐caspase inhibitor reduced apoptosis but showed weaker inhibition of cell proliferation than the autophagy inhibitor. We concluded that p53‐dependent cell death in SF126 cells comprises caspase‐dependent and caspase‐independent apoptosis and autophagic cell death, and the induction of autophagy as well as apoptosis could be a new strategy to treat some type of WT p53‐retaining tumors. (Cancer Sci 2011; 102: 799–807)

18 F-fluorodeoxyglucose specimen-positron emission mammography delineates tumour extension in breast-conserving surgery: Preliminary results

ObjectivesWe aimed to determine whether high-resolution specimen-positron emission mammography (PEM) using fluorodeoxyglucose (18F-FDG) can reveal extension of breast cancer in breast-conserving surgery (BCS), and assess the safety of radiation exposure to medical staff.MethodsSixteen patients underwent positron emission tomography, and then BCS with intraoperative frozen section analysis on the same day. Resected specimens with remaining 18F-FDG accumulation were scanned by high-resolution PEM. At least 1 day after surgery, tumour extension was evaluated by three independent experienced readers and by binarized images from the specimen-PEM data. Intraoperative exposure of medical staff to 18F-FDG was measured.ResultsSpecimen-PEM evaluations of binarized images and the three investigators detected all (100 %, 12/12) invasive lesions and 94.4 % (17/18) of in situ lesions using both methods. The positive predictive value of the accumulated lesions was 74.4 % (29/39) for the binarized images and 82.9 % (29/35) for the three investigators. Analysis of intraoperative frozen sections detected 100 % (2/2) of the margin-positive cases, also detected by both specimen-PEM evaluation methods with no false-positive margin cases. The mean exposure of the medical staff to 18F was 18 μSv.ConclusionsSpecimen-PEM detected invasive and in situ lesions with high accuracy and allowable radiation exposure.Key points• Specimen-PEM detected invasive and in situ lesions with high accuracy.• Specimen-PEM predicted complete resection with the same accuracy as frozen section analysis.• Breast-conserving surgery after fluorodeoxyglucose injection was performed with low medical staff exposure.

Increased centrosome number in BRCA-related breast cancer specimens determined by immunofluorescence analysis

BRCA‐related breast carcinoma can be prevented through prophylactic surgery and an intensive follow‐up regimen. However, BRCA genetic tests cannot be routinely performed, and some BRCA mutations could not be defined as deleterious mutations or normal variants. Therefore, an easy functional assay of BRCA will be useful to evaluate BRCA status. As it has been reported that BRCA functions in the regulation of centrosome number, we focused on centrosome number in cancer tissues. Here, 70 breast cancer specimens with known BRCA status were analyzed using immunofluorescence of γ‐tubulin (a marker of centrosome) foci. The number of foci per cell was higher in cases with BRCA mutation compared to wild‐type cases, that is, 1.9 (95% confidence interval [CI], 1.5‐2.3) vs 0.5 (95% CI, 0.2‐0.8) (P < .001). Specifically, foci numbers per cell in BRCA1 and BRCA2 mutation cases were 1.2 (95% CI, 0.6‐1.8) and 2.2 (95% CI, 1.7‐2.6), respectively, both higher than those in wild‐type cases (P = .042 and P < .0001, respectively). The predictive value of γ‐tubulin foci as determined by area under the curve (AUC = 0.86) for BRCA status was superior to BRCAPRO (AUC = 0.69), Myriad Table (AUC = 0.61), and KOHBRA BRCA risk calculator (AUC = 0.65) pretest values. The use of γ‐tubulin foci to predict BRCA status had sensitivity = 83% (19/23), specificity = 89% (42/47), and positive predictive value = 77% (20/26). Thus, γ‐tubulin immunofluorescence, a functional assessment of BRCA, can be used as a new prospective test of BRCA status.

Abstract P1-02-12: High-resolution specimen-positron emission mammography (s-PEM) indicates the spread of cancer in breast-conserving surgery

Background: Breast cancer surgery, including breast-conserving surgery or sentinel lymphnode biopsy, has become minimally invasive. Recent consensus guidelines on the margins for breast-conserving surgery have defined a negative margin as one with no ink on tumor, regardless of the distance from the tumor, whereas a positive margin has ink on the tumor. Therefore, the extent of required tumor resection will decrease for cosmoses and expand the indications for breast-conserving surgery. Therefore, there is a real need for a more accurate imaging of tumor spread. In particular, a functional imaging of breast cancer specimens will help determine negative margins in breast-conserving surgery. We collaborated with Sendai Medical Imaging Center (SMIC) who have high-resolution positron emission mammography (PEM) with the novel scintillator Pr3+-doped transparent ceramic Lu3Al5O12 (Pr:LuAG) for the imaging of breast cancer specimen with 18F-fluodeoxyglucose (FDG). Methods: With the approval of the hospital ethics committee, positron emission tomography (PET) was conducted preoperatively on the day of surgery with 18F-FDG. After the PET results were explained to the patients, breast-conserving surgery was performed in 11 patients from February to July 2014. In the operating room, medical staff exposures were calculated with a portable dosimeter. All of the cases were carried out intraoperative frozen sections that were taken from all sides of the outside of the specimens. The specimens were serially sectioned in 5-mm slices for permanent histology. After the specimens were removed, they were sent to the SMIC and high-resolution specimen-PEM (s-PEM) was conducted. We evaluated the detection rate of s-PEM in in situ or invasive lesions (extent of locations were tolerated up to a 10% error), the predictive value of margin status between s-PEM and frozen section analysis and medical stuff exposure. Results: In 11 breast specimens, eight invasive lesions and 15 in situ lesions (excluding the foci that exist in less than three carcinoma ducts) were confirmed by permanent histology. s-PEM detected all of the invasive lesions and 14 out of 15 in situ lesions. Three breast specimens had positive margins in permanent histology. Two out of three cases with positive margin accumulated 18F-FDG at the edge of the specimens, which were considered positive margins with s-PEM, whereas positive margins were only one out of three of the frozen section analysis. In contrast, a false positive margin was found in only one case with s-PEM, which confirmed the preoperative core-needle biopsy that was diagnosed as an intraductal proliferative lesion. Medical staff exposure was determined for the operator, first assistant, second assistant, anesthesiologist, scrab nurse and circulating nurse as 31, 34, 25, 11, 21 and 6 µSV, respectively. Conclusion: s-PEM detected not only invasive lesions but also in situ lesions with great accuracy. It might accelerate the decrease in the extent of resection required in breast-conserving surgery. However, further studies are needed to elucidate the characteristics of the false positive cases in s-PEM. Citation Format: Gou Watanabe, Masatoshi Itoh, Dan Kyokutou, Mika Watanabe, Takanori Ishida, Akihiko Suzuki, Minoru Miyashita, Hiroshi Tada, Naoko Mori, Noriaki Ohuchi. High-resolution specimen-positron emission mammography (s-PEM) indicates the spread of cancer in breast-conserving surgery [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-02-12.

Abstract P2-10-43: The combination of immunohistochemistry for predictingTP53mutation is useful prognostic marker in breast cancer.

The comprehensive expression analysis classified subtypes of breast cancer which is clinically applied for immunohistochemistry (IHC) with ER, HER2 and Ki67. On the other hand, recent whole genome sequencing reveals a certain quantity of TP53 mutation in breast cancer, especially with ER negative tumor. Therefore, it is important for risk evaluation that TP53 mutation is applied for IHC. However, p53 IHC is not well correlated with TP53 mutation because of false negative (deletion mutant) and false positive (stabilization of p53). This study is initiated to improve accuracy to combine the IHC of p53 and p53-regulated protein for predicting TP53 mutation. First, we explored p53-dependent down regulated proteins to rescue the false negative of p53 IHC cases. Using the microarray analysis and DNA sequencing of TP53 gene in 37 breast cancer patients, we extracted 97genes which were overexpressed in TP53 mutation breast cancer cases compared with TP53 wild cases. Furthermore, using the microarray analysis by two tet-inducible p53 cell lines, 597 genes were extracted for p53-dependent repressed genes. There were 29 common genes between both of them. In 29genes, we picked up three genes, BIRC5, BUB1 and PLK1 which were confirmed with p53-dependent repression by RT-PCR or luciferase assay. Next, the correlation of TP53 status and IHC of these genes products and p53 were evaluated. In 37 breast cancer patients, TP53 mutations were detected in 19 cases (51%). There were 11 missense mutations, 5 protein-truncating mutations and two splicing-site mutations. p53 IHC positive (>10%) were 12 cases (32.4%) which included only 9 TP53 mutations. Five protein-truncating TP53 mutations were all negative staining of p53. Among BIRC5, BUB1 and PLK1 proteins, positive staining of PLK1 is the most correlated with TP53 mutations, sensitivity 0.78, specificity 0.84 and positive predictive value 0.78 (p Therefore IHC of p53 + and p21 – (to decrease false positive of p53) or p53- and PLK1+ (to save false negative of p53) were defined as IHC-TP53 mutation+. The IHC-TP53 mutation + were strongly correlated with TP53 mutations, sensitivity 0.89, specificity 0.95 and positive predictive value 0.94 (p The combination of p53, p21 and PLK1 IHC are well predicted TP53 mutations and is a useful biological, prognostic marker such as ER or HER2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-43.