Investigation of Ki67 and Phospho-Histone H3 Expressions in Urothelial Carcinoma of the Bladder by Immunohistochemical Method.

BACKGROUND: In our study, it is aimed to investigate the relationship between Ki67 and phospho-histone H3 (pHH3) expressions in bladder urothelial carcinomas, with clinicopathological parameters and survival, which have prognostic value. METHODS: The study included 44 cases of high-grade urothelial carcinoma (HGUC), 37 cases of low-grade urothelial carcinoma (LGUC), and 11 nontumoral bladder cases. Ki67 and pHH3 were applied to the paraffin blocks of the tissues of 81 urothelial carcinoma and 11 nontumoral bladder cases by immunohistochemical method. Percentages of Ki67 and pHH3 expressions were evaluated by digital imaging analysis method. Expression percentages were compared with various clinicopathological parameters, and the relationship between them was evaluated. RESULTS: Ki67 was expressed in 28% of urothelial carcinoma cases and 1% of nontumoral cases. pHH3 was expressed in 10.32% of urothelial carcinoma cases and 0.16% of nontumoral cases. In our study, we found significantly higher Ki67 and pHH3 expressions in urothelial carcinoma compared to nontumoral cases. There was a statistically significant relationship (p < 0.05) and a positive correlation between Ki67 expression and lymphovascular invasion, pT stage, and histological grade. A statistically significant relationship (p < 0.05) and a positive correlation were found between pHH3 expression and lymphovascular invasion, pT stage, recurrence, and histological grade. In addition, a statistically significant relationship was found between Ki67 and pHH3 expressions. In our study, survival was found to be low in high-grade urothelial carcinoma cases with lymphovascular invasion, advanced age (65 years and older), and high Ki67 and pHH3 expression rates. CONCLUSIONS: According to our findings, high Ki67 and pHH3 expressions were found to be associated with poor prognostic parameters such as advanced pathologic stage, high histologic grade, and low survival. Our findings suggest that Ki67 and pHH3 may play a role in the differentiation, progression, and aggressive behavior of urothelial carcinoma. However, further studies are needed to confirm our findings and determine the role of these markers in urothelial carcinoma.

The Prognostic Effect of HER2 Gene Amplification in High-Grade Endometrial Carcinomas and its Correlation with Protein Overexpression.

Introduction. New therapeutic agents and biomarkers are needed for the treatment of aggressive endometrial cancer subtypes. Recently, HER2 has been recommended to be tested routinely in serous endometrial cancers. The aim of this study is to investigate the correlation between HER2 (ERBB2) protein overexpression and HER2 gene amplification and the relationship of HER2 gene amplification with prognosis in cancers with serous morphology. In addition, the concordance of HER2 testing in paired curettage and hysterectomy specimens is also investigated. Methods. Twenty five serous carcinomas and 8 carcinosarcomas with a serous morphology were included in the study. HER2 staining was performed on whole tissue sections by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). The system, which was proposed by Fader et al was used to evaluate the stainings. Results. Protein overexpression was detected in 27.3% (n = 9) of the cases, and gene amplification in 30.3% (n = 10). A significant positive correlation was found between the two methods (P < .0001). HER2 IHC revealed a heterogeneous staining pattern, such as intense complete membranous in solid areas, and basolateral in papillary and glandular areas. HER2 gene amplification was significantly associated with shorter overall (P = .005) and disease-free (P = .014) survival. The concordence of the results in curettage and hysterectomy specimens was also significantly high. Conclusion. HER2 is an important prognostic and predictive marker for endometrial cancers with serous morphology. HER2 IHC/ISH testing can be performed by using diagnostic curettage specimens which contain enough viable tumor cells. However, pathologists should be aware of the intratumoral heterogeneity for HER2 staining.

Effect of corticosteroid (triamcinolone acetonide) and chlorhexidin on chemotherapy- induced oxidative stress in buccal mucosa of rats.

Oral mucositis (OM) refers to erythematous and ulcerative lesions of the oral mucosa. This pathology can occur by various causes. Cancer therapy is one of the well-known causes of OM such as chemotherapy and/or with radiation therapy. It has been widely mentioned that oxidative stress parameters such as lipid peroxidation (LP) levels increase during cancer process. Glutathione (GSH) is one of the major intracellular enzymes to detoxify oxidant molecules. The aim of this study was to investigate and compare the effects of Triamcinolone Acetonide (TA), a synthetic steroid chlorhexidine (CHX), a chemical antiseptic, on 5- fluorouracil (5-FU), a chemotherapeutic agent and soft abrasion induced OM in buccal mucosa of rats.OM was induced in rats through a combination of 5-FU treatment and mild abrasion of the cheek pouch with a wire brush. Buccal mucosa lipid peroxidation (LP) levels were higher (p< 0.05) in 5-FU group than in control although LP levels were lower (p<0.05) in TA group than in control group. The reduced glutathione levels were lower (p<0.05) in 5-FU group than in the control group although its level was higher (p<0.05) in TA and CHX groups than in the 5-FU group. Glutathione peroxidase activity was also higher (p<0.05) in TA group than the 5- FU group. In histopathological analyses, treatment with TA reduced 5-FU induced inflammatory cell infiltration and ulceration (p<0.001) but not with CHX.In conclusion, we observed that TA and CHX treatment modulated chemotherapy induced oxidative injury in the rat OM. However, only TA histopathologically ameliorated the 5-FU induced OM of rats. These findings suggest that TA is a useful agent for management of experimental oxidative injury and OM caused by the chemotherapy.

Expression of NGF, GDNF and MMP-9 in prostate carcinoma.

The aim of the present study was to investigate the immunohistochemical expression of NGF, GDNF and MMP-9 in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PC), and to analyse their association with the clinicopathological parameters in PC cases. Immunohistochemistry was performed on the tissue microarray (TMA) sections of 30 BPH, 40 HGPIN and 121 primary PC tissues. There was a significant difference regarding the expression of NGF and GDNF between PC and HGPIN (p<0.0001; p<0.0001), and PC and BPH (p=0.001; p<0.0001), but not between HGPIN and BPH (p>0.05). Furthermore MMP-9 expression was significantly different among all groups (PC vs. HGPIN, p<0.0001; PC vs. BPH, p<0.0001; HGPIN vs. BPH, p=0.001). NGF, GDNF and MMP-9 expression was significantly stronger in cases with high Gleason score (p<0.0001, p=0.004, p<0.0001 respectively) and pT stage (p=0.046, p=0.004, p=0.001, respectively) in PC cases. All these markers were also associated with perineural, lymphovascular and extraprostatic invasion (p <0.05). In addition, a positive correlation was found between NGF and MMP-9 (p<0.0001, r=0.435), NGF and GDNF (p<0.0001, r=0.634), and GDNF and MMP-9 (p<0.0001, r=0.670) in PC cases. According to our results we suggest an interaction between NGF, GDNF and MMP-9 during the transition to malignancy in PC. Also this interaction may involve in regulating PC cell differentiation, tumor invasion, progression, and the agressiveness of PC.

Effect of a corticosteroid (triamcinolone) and chlorhexidine on chemotherapy-induced oxidative stress in the buccal mucosa of rats.

Oral mucositis manifests as erythematous and ulcerative lesions of the oral mucosa. Among its various causes, cancer treatment (e.g., chemotherapy with or without radiation therapy) is one of the more well known. It has been widely mentioned that oxidative stress parameters such as lipid peroxidation levels increase during the cancer process. Glutathione is one of the major intracellular enzymes used to detoxify oxidant molecules; it exists in both a reduced and oxidized state. Reduced glutathione is used as a substrate to synthesize glutathione peroxidase. We conducted a study to investigate and compare the effects of triamcinolone (a synthetic steroid) and chlorhexidine (a chemical antiseptic) on 5-fluorouracil (5-FU; a chemotherapeutic agent)-induced oral mucositis in the buccal mucosa of 36 rats. Oral mucositis was induced through a combination of 5-FU treatment and mild abrasion of the cheek pouch with a wire brush. The rats were treated with one of four regimens: saline placebo (group I), 5-FU only (group II), 5-FU plus triamcinolone (group III), and 5-FU plus chlorhexidine (group IV). Three rats in the triamcinolone group died of unknown causes on days 7 and 8, and 3 rats in the chlorhexidine group died on days 7 and 9. On day 9, the remaining 30 rats were sacrificed and examined. Buccal mucosa lipid peroxidation levels were significantly higher in the 5-FU-only group than in the control group and significantly higher in the control group than in the triamcinolone group (p < 0.05 for both). Levels of reduced glutathione were significantly lower in the 5-FU-only group than in both the triamcinolone group and the chlorhexidine group (p < 0.05). Glutathione peroxidase activity was significantly higher in the triamcinolone group than in the 5-FU-only group (p < 0.01). Histopathologic analysis revealed that treatment with triamcinolone significantly reduced 5-FU-induced inflammatory cell infiltration and ulceration (p < 0.001); no such reduction was seen with chlorhexidine. In conclusion, we observed that triamcinolone and chlorhexidine treatment modulated chemotherapy-induced oxidative injury in rat oral mucositis. However, only triamcinolone histopathologically ameliorated 5-FU-induced oral mucositis. These findings suggest that triamcinolone is a useful agent for the management of experimental oxidative injury and oral mucositis caused by 5-FU chemotherapy.

The role of immunohistochemical adrenomedullin and Bcl-2 expression in development of type-1 endometrial adenocarcinoma: Adrenomedullin expression in endometrium.

BACKGROUND: Adrenomedullin (AM) is a pluripotent peptide first discovered from human pheochromocytoma. AM expression has been shown in various cancer types including endometrium cancer. Bcl-2 is an antiapoptotic protein which might be regulated by AM in hypoxic conditions. The aim of the present study was to investigate the role of AM and Bcl-2 expressions in carcinogenesis of type-1 endometrium cancer. MATERIALS AND METHOD: Study group consisted of 10 proliferative endometrium, 22 simple endometrial hyperplasia, 23 endometrial intraepithelial neoplasia (EIN) and 30 Grade 1 endometrioid adenocarcinoma patients. AM and Bcl-2 expressions were investigated by immunohistochemistry. RESULTS: Mean AM Allred score was 3±2.6, 5.6±1.6 and 5.7±2.5 in benign, EIN and adenocarcinoma groups, respectively. AM expression was significantly higher in EIN and adenocarcinoma groups than in benign endometrium group (p<0.05). Mean Bcl-2 Allred score was 6.4±2.1, 5.2±2.6, 2.3±2 in benign endometrium, EIN and adenocarcinoma groups, respectively. Mean Bcl-2 Allred score was similar between benign endometrium and EIN groups (p>0.05). However, it was significantly lower in adenocarcinoma group (p<0.05). An inverse correlation between AM and Bcl-2 expressions was found (r: -0.4, p<0.001). CONCLUSIONS: Our findings showed that AM expression increased in progression from benign endometrium to EIN and type-1 adenocarcinoma while expression of Bcl-2 decreased in transition from EIN to carcinoma.

L-carnitine reduces acute lung injury in experimental biliary obstruction.

OBJECTIVES: To investigate the protective effects of L-carnitine (LC) on lungs in an experimental obstructive jaundice (OJ) model. METHODS: This was conducted for 2 months between May 2011 and July 2011 at Suleyman Demirel University School of Medicine Experimental and Clinical Research Center, Isparta, Turkey. Thirty-eight Wistar-Albino rats with an average weight of 250-300 g were divided into 3 groups of control, OJ, and OJ + L-carnitine treatment (LCT). L-carnitine was injected intravenously into the tail vein at a dose of 50 mg/kg/day for 10 days to the LCT group. Animals were sacrificed 10 days later. Enzyme levels were measured in the lung tissue; malondialdehyde, myeloperoxidase (MPO), glutathione peroxidase (GSH-Px), catalase, and superoxide dismutase. Tumor necrosis factor-alfa, interleukin 6 (IL-6), IL-8, and C-reactive protein levels were studied in plasma samples. Histopathological changes in the lungs were examined.  RESULTS: There was a decreased in GSH-Px, MPO, and IL-8 levels (p less than 0.05) in the LCT group. The histopathological examination showed that neutrophil leukocyte infiltration and edema formation decreased and destruction of lung parenchyma disappeared following the treatment with LC (p less than 0.05).  CONCLUSION: L-carnitine has a protective effect against lung damage due to experimental obstructive jaundice, possibly by altering anticytokine and antioxidant activity, and by decreasing the neutrophil migration.

The clinicopathological and prognostic significance of CD24, CD44, CD133, ALDH1 expressions in invasive ductal carcinoma of the breast: CD44/CD24 expression in breast cancer.

BACKGROUND: Recently, there are several studies about cancer stem cells (CSC), indicating that they are the cells that initiate the tumor, provide progression, metastasis and responsible for the aggressive tumor behavior. MATERIALS AND METHODS: The purpose of this study is to investigate the expressions of CD24, CD44, their different combinations, ALDH1 and CD133 in invasive ductal carcinoma. Their relationships with clinicopathologic parameters, such as tumor grade, lymphovascular invasion, tumor size, axillary lymph node involvement, stage, hormone receptors, HER2 expression, basal like tumors, triple negative status and prognosis were also investigated. Tissue microarray method was used to investigate immunohistochemical CD24, CD44, ALDH1 and CD133 expressions in 105 invasive ductal carcinoma cases. RESULTS: CD133 expression was significantly associated with tumor size (p=0.023) and stage (p=0.009). CD133 expression was decreased in tumors with larger tumor size, higher stage and lymphovascular invasion. CD133 expression was positively correlated with CD44 (r=0.212, p=0.032) and CD44(+)/CD24(+) (r=0.202, p=0.040) expressions. CD44, CD24 and ALDH1 expressions showed no significant relationship and correlation with clinicopathologic features. There was a significant relationship (p=0.048) between CD44(+)/CD24(-/low) phenotype and basal like tumors. EGFR expression was positively correlated with CD44(+)/CD24(-/low) phenotype (r=0.211, p=0.036). CONCLUSIONS: Basal like tumors are enriched for CSCs with CD44(+)/CD24(-/low) phenotype. CD133 can detect a different population of CSC in breast carcinoma.

Protective effect of theophylline on renal functions in experimental pneumoperitoneum model.

Our objective in this experimental study is to research the effect of the intra-abdominal pressure which rises following pneumoperitoneum and whether Theophylline has a possible protective activity on this situation. In our study, 24 Wistar Albino rats were used. Rats were divided into two groups. The first group was set for only pneumoperitoneum model. The second group was given 15 mg/kg of Theophylline intraperitoneally before setting pneumoperitoneum model. Then urea, creatinine, cystatin-C, tissue and serum total antioxidant capacity, total oxidant capacity and oxidative stress index in two groups were measured and compared with each other. Apoptosis and histopathological conditions in the renal tissues were examined. The differences between the groups were analyzed with the Mann-Whitney U test. Results were considered significant at p < 0.05. No statistically significant difference was determined between tissue and serum averages in two groups in terms of TAS, TOS and OSI values (p > 0.05). The mean value of urea were similar in pneumoperitoneum and pneumoperitoneum + theophylline groups (p = 0.12). The mean cystatin-C value was 2.2 ± 0.3 µg/mL in pneumoperitoneum, 1.74 ± 0.33 µg/mL in pneumoperitoneum + theophylline (p = 0.002). According to our study, lower cystatin-C levels in the group, where Theophylline was given, are suggestive of lower renal injury in this group. However, this opinion is interrogated as there is no difference in terms of tissue and serum TAS, TOS, OSI and urea values between the groups.

The relation of beclin 1 and bcl-2 expressions in high grade prostatic intraepithelial neoplasia and prostate adenocarcinoma: a tissue microarray study.

The aim of the present study was to evaluate the expressions of beclin 1 and bcl-2 in prostate cancer (PC) and high grade prostatic intraepithelial neoplasia (HGPIN), and to investigate their relationship with clinicopathological parameters. The study included 30 benign prostatic hyperplasia (BPH), 40 HGPIN and 106 primary PC cases. The expressions of beclin 1 and bcl-2 were assessed semiquantitatively based on both the percentage and intensity of positive staining cells. Beclin 1 was positive in 27 (90%) BPH, 37 (92.5%) HGPIN, and 90 (84.9%) PC cases (p>0.05). Bcl-2 immunostaining was detected in 99 (93.4%) PC, 37 (92.5%) HGPIN, and 9 (30%) BPH cases (p<0.0001). Regarding expression scores, beclin 1 was significantly lower in PC cases than in the HGPIN and BPH groups (p<0.0001), and it was also negatively correlated with Gleason score (p=0.004, r=-0.274). Bcl-2 expression score was significantly higher in PC than in the other groups (p<0.0001), and also positively correlated with Gleason score (p<0.0001, r=0.425). Furthermore, a negative correlation was found between bcl-2 and beclin 1 expression scores in PC cases (p=0.006, r=-0.265). Our results suggest an association between bcl-2 and beclin 1 expressions in malignant transformation of prostate tissue and also in regulating PC cell differentiation, progression and the aggressiveness of PC.

Corneal limbal sebaceous adenoma.

PURPOSE: The aim of this study was to describe a case of a corneal limbal sebaceous adenoma. METHODS: An 83-year-old male patient presented with a white solid nodular lesion covering most of the cornea of his right eye. The corneal lesion was removed totally. The corneal surface was reconstructed by amniotic membrane transplantation. RESULTS: The histopathological examination of the lesion revealed the presence of a sebaceous adenoma. A systemic examination revealed no other abnormalities. CONCLUSIONS: Sebaceous adenomas on the ocular surface are known to be very rare. To the best of our knowledge, this is the first case of a sebaceous adenoma located on the cornea, reported in the literature. It is of vital importance to make a differential diagnosis of sebaceous adenomas of the eyelid and conjunctiva with the Muir-Torre syndrome associated with visceral malignancies.

The correlation between preputial blood flow and microvessel density in distal hypospadias: a prospective clinical study.

OBJECTIVE: A prospective clinical study was designed to investigate the correlation between preputial blood flow (BF) and microvessel density (MVD). PATIENTS AND METHODS: A total of 44 children were included in the study. The hypospadias group consisted of 16 children undergoing distal hypospadias repair, and the control group consisted of 28 age-matched healthy children undergoing circumcision. BFs were measured using a laser Doppler flowmeter on the most distal part of the dorsal prepuces, and then the tissue samples were harvested from the same location. They were immunostained with an antibody against CD31 in order to assay MVD. The statistical analyses were carried out using Student's t test and Pearson's correlation analysis. RESULTS: The preputial MVD was found to be significantly decreased in the patients with hypospadias compared with the healthy children (33.95 ± 9.79 vs. 48.25 ± 10.08; p < 0.05), whereas there was no difference in terms of the BF (40.58 ± 16.16 vs. 33.09 ± 19.65; p > 0.05). CONCLUSIONS: We found no correlation between the preputial MVD and BF in the present study. This result suggests that reduced preputial MVD does not have any influence on BF in distal hypospadias.

Beclin 1 and bcl-2 expressions in bladder urothelial tumors and their association with clinicopathological parameters.

Beclin 1 plays a critical role in the regulation of autophagy, apoptosis, differentiation, as well as in the development and progression of cancer. The aim of this study was to examine the expression of beclin 1 and bcl-2 in bladder urothelial tumors, and to investigate the relationship between these two markers and clinicopathological parameters. Our study included 84 bladder urothelial tumors and 10 non-tumoral bladder tissues. Immunohistochemistry was performed on tissue microarray (TMA) sections and was evaluated semiquantitatively on the basis of the percentage of positively stained cells (proportion) and staining intensity. A significant association was found between the expression score of beclin 1 and pT stages of the urothelial tumors (p=0.012). Also, the level of beclin 1 expression inversely correlated with histological grade and pT stages (p=0.009, r=-0.284; p=0.001, r=-0.361, respectively). The bcl-2 expression level positively correlated with histological grade and pT stages of the urothelial tumors (p=0.026, r=0.243; p<0.0001, r=0.491, respectively). In addition, the level of beclin 1 expression tended to be inversely correlated with the bcl-2 expression level in urothelial tumors (p=0.055, r=-0.210). According to our data, down-regulation of beclin 1 expression and also bcl-2 overexpression seem to play an important role in the progression and aggressiveness of bladder urothelial tumors.

[Coexistence of multiple gastrointestinal stromal tumors and signet ring cell carcinoma of stomach in a patient with neurofibromatosis type-1: case report]. / Nörofibromatozis Tip-1'li Hastada Multipl Gastrointestinal Stromal Tümör ve Midede Tasli Yüzük Hücreli Karsinom Birlikteligi: Olgu Sunumu.

Gastrointestinal stromal tumors are mesenchymal neoplasias which are derived from Cajal's interstitial cells. The most common site of involvement is the stomach. It may be multiple in patients with Neurofibromatosis Type-1, while the small intestine is the most common location. In this case report, we aimed to present a Neurofibromatosis Type-1 patient, showing coexistence of multiple gastrointestinal stromal tumors in the stomach and small intestine with a signet ring cell carcinoma in the stomach. A 74-year-old female patient with poor appetite, vomiting and stomach ache was admitted to the hospital. After detection of a tumoral lesion with an ulcerated surface in stomach during the upper gastrointestinal tract endoscopic examination, the patient underwent surgery. During the operation, multiple nodular lesions were observed in the serosal surfaces of the small intestine and stomach. Gastrectomy and partial small intestine resection specimens were evaluated and the patient was diagnosed as signet ring cell carcinoma in the stomach, and multiple gastrointestinal stromal tumors in the serosal surfaces of both the stomach and small intestine. Resection specimens of patients with GIST need to be evaluated carefully on macroscopic examination, considering the possible presence of a coexistent tumoral lesion.

Increased expression of COX-2 in recurrent basal cell carcinoma of the skin: a pilot study.

INTRODUCTION: Basal cell carcinoma (BCC) is the most frequent malignant skin tumor. BCC rarely metastasizes, but it is often locally aggressive. Cyclooxygenase-2 (COX-2) is critical for tumor formation, angiogenesis and metastasis. Matrix metalloproteinases (MMPs) are the members of the family of zinc (Zn)- and calcium-dependent endopeptidases that degrade the extracellular matrix. MATERIALS AND METHODS: In our study, we used immunohistochemical methods for the evaluation of COX-2, MMP-2 and MMP-9 expression in tissue samples of 30 primary and 10 recurrent skin BCC cases. RESULTS: Immunohistochemical COX-2 expression was significantly higher in the infiltrating pattern of BCC compared with the nodular (P = 0.005) and superficial (P = 0.041) subtypes in the primary BCC group. There was not a significant difference between nodular and superficial BCCs for COX-2 expression. In addition, COX-2 expression was significantly higher in the recurrent BCC group than in the primary BCC group (P = 0.030). There was no statistically significant difference between the histological subtypes of primary BCCs and between primary and recurrent BCCs for MMP-2 and MMP-9 expressions. CONCLUSIONS: Our data confirm previous findings that COX-2 and MMP-9 expressions are increased in BCC. Our results revealed an elevated COX-2 expression in recurrent BCCs. We suggest that COX-2 inhibition might have beneficial effects in BCCs, especially for the tumors with a higher level of COX-2 expression or aggressive phenotype.

Immunohistochemical COX-2 overexpression correlates with HER-2/neu overexpression in invasive breast carcinomas: a pilot study.

Cyclooxygenase-2 (COX-2) is a prostaglandin synthase that catalyzes the synthesis of prostaglandin G2 and H2. It has been shown that COX-2 plays an important role in tumorigenesis of different tumor types and it is thought to take part in breast carcinogenesis. In the present study, we aimed to investigate the relationship of immunohistochemical COX-2 expression with clinicopathological parameters, including HER-2/neu overexpression in invasive breast carcinoma (IBC). Our study population comprised 10 normal breasts, 25 ductal carcinomas in situ (DCIS), and 51 invasive breast carcinomas. Immunohistochemical overexpressions of COX-2 and HER-2/neu were investigated in sections of formalin-fixed, paraffin-embedded blocks by 3 observers. In normal breast, DCIS and IBC, the COX-2 overexpression rate was 0%, 84%, and 58.8%, respectively. In IBC, COX-2 overexpression had a significant relationship with HER-2/neu overexpression (p=0.026) and a high histological grade (p=0.026). COX-2 expression in both DCIS (n=25) and IBC (n=51) was significantly higher than in normal breast tissue (p<0.0001). In addition, the COX-2 expression rate was significantly higher in DCIS than in IBC (p=0.042). Our results indicated that COX-2 overexpression correlates with aggressive phenotypic features, such as HER-2/neu overexpression and high histological grade in IBC. Increased expression of COX-2 in both DCIS and IBC in comparison to normal breast could indicate a role in breast carcinogenesis. COX-2 overexpression may provide a clinically useful biomarker for estimating tumor aggressiveness.