TERT promoter mutations are frequent and show association with MED12 mutations in phyllodes tumors of the breast

Background:

Phyllodes tumors are rare fibroepithelial neoplasms of the breast, which carry the potential risk of local recurrence and metastasis. Phyllodes tumors share several histological features with fibroadenomas, and no widely accepted markers for distinguishing these lesions have been identified.

Methods:

We analyzed molecular abnormalities related to telomere elongation in tumors, including TERT promoter mutations, as well as loss of expression of ATRX and DAXX, in a total of 104 phyllodes tumors and fibroadenomas.

Results:

Sequencing analyses showed that TERT promoter mutations were frequent in phyllodes tumors (30/46, 65%), but rare in fibroadenomas (4/58, 7%). Among phyllodes tumors, the mutations were more frequent in borderline tumors (13/15, 87%), but were also common in benign (9/18, 50%) and malignant tumors (8/13, 62%). Remarkably, all but one TERT promoter-mutated tumor also contained MED12 mutations, indicating that these mutations are strongly associated (P=8.4 × 10⁻⁶). Expression of ATRX and DAXX, as evaluated by immunohistochemistry, was retained in all tumors.

Conclusions:

Our observations suggest a critical role of TERT promoter mutations, in cooperation with MED12 mutations, in the development of phyllodes tumors. Because TERT promoter mutations are rare among fibroadenomas, their detection may be of potential use in discriminating between phyllodes tumors and fibroadenomas.     

Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations

Clinical and experimental studies have suggested that tumour hypoxia is associated with poor treatment outcome and that loss of apoptotic potential may play a role in malignant progression of neoplastic cells. The tumour suppressor gene p53 induces apoptosis under certain conditions and microenvironmental tumour hypoxia may select for mutant tumour cells with diminished apoptotic potential due to lack of p53 function. The aim of this study was to evaluate the prognostic relevance of oxygenation status for treatment outcome and to compare pre-treatment tumour oxygenation measurements were done in 31 of those by PCR using DNA extracted from paraffin-embaedded sections (n = 2) or frozen biopsies (n = 29). The overall median of the tumour median pO(2)was 19 mmHg (range 1-58 mmHg). Only 6 tumours had functional p53 mutations and no association was found between mutant p53 and tumour hypoxia. Five out of 6 STS with lower histopathological grade were well-oxygenated whereas high-grade STS were both hypoxic and well-oxygenated. At a median follow-up of 74 months, 16 patients were still alive among 28 available for survival analysis. When stratifying into hypoxic and well-oxygenated tumours patients with the most hypoxic tumours has a statistically poorer disease-specific and overall survival at 5 years. In conclusion hypoxia was an indicator for both a poorer disease specific and overall survival in human STS but hypoxic tumours were not characterized by mutations in the p53 gene.     

PIK3CA mutations are frequently observed in BRCAX but not BRCA2 -associated male breast cancer

Introduction

Although a substantial proportion of male breast cancers (MBCs) are hereditary, the molecular pathways that are activated are unknown. We therefore examined the frequency and clinicopathological associations of the PIK3CA/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and their regulatory genes in familial MBC.

Methods

High resolution melting analysis and confirmatory sequencing was used to determine the presence of somatic mutations in PIK3CA (exon 9 and 20), AKT1 (exon 4), KRAS (exon 2) and BRAF (exon 15) genes in 57 familial MBCs. Further analysis of the PIK3CA/mTOR pathway was performed using immunohistochemistry for the pAKT1, pS6 and p4EBP1 biomarkers.

Results

PIK3CA somatic mutations were identified in 10.5% (6 of 57) of cases; there were no AKT1, KRAS or BRAF somatic mutations. PIK3CA mutations were significantly more frequent in cancers from BRCAX patients (17.2%, 5/29) than BRCA2 (0%, 0/25) carriers (P = 0.030). Two BRCAX patients had an E547K mutation which has only been reported in one female breast cancer previously. PIK3CA mutation was significantly correlated with positive pS6 (83.3% vs. 32.0%, P = 0.024) and negative p4EBP1 (100% vs. 38.0%, P = 0.006) expression, but not pAKT expression. Expression of nuclear p4EBP1 correlated with BRCA2 mutation carrier status (68.0% vs. 38.7%, P = 0.035).

Conclusions

Somatic PIK3CA mutation is present in familial male breast cancer but absent in BRCA2 carriers. The presence of two of the extremely rare E547K PIK3CA mutations in our cohort may have specific relevance in MBCs. Further study of PIK3CA in MBCs, and in particular BRCAX patients, may contribute to further establishing the relevance of specific PIK3CA mutations in MBC aetiology and in the identification of particular patient groups most likely to benefit from therapeutic targeting with the novel PIK3CA inhibitors that are currently in development.     

Quantitative analysis of aromatase, sulfatase and 17beta-HSD(1) mRNA expression in soft tissue metastases of breast cancer

Expression of the estrogen-synthesizing genes aromatase, steroid sulfatase (STS) and 17beta-hydroxysteroid dehydrogenase type1 (17beta-HSD(1)) has been shown to be up-regulated in primary breast cancer tissue but their expression status in metastatic tumor tissue has yet to be determined. The mRNA expression levels of the three estrogen-synthesizing genes as well as of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and cyclooxygenase (COX)-2, all of which have been reported to up-regulate the estrogen-synthesizing genes, were determined by means of a real-time PCR assay in 100 primary breast cancer tissues and 15 soft tissue metastases. In addition, PCR-gel electrophoresis was used to determine the proportion (%) of promoter (l.4, l.3, Pll and l.7) usage of aromatase. Aromatase and STS mRNA levels were significantly (P=0.04 and P=0.03, respectively) higher in soft tissue metastases than in primary tumors, while 17beta-HSD(1) mRNA levels tended (P=0.09) to be higher. The proportions of the promoter usages were very similar for primary tumors and soft tissue metastases, and the mRNA levels of TNF-alpha, IL-6 and COX-2 were not significantly different. Levels of aromatase, STS and 17beta-HSD(1) mRNA are up-regulated in soft tissue metastases compared to those in primary tumors, suggesting that intra-tumoral estrogen synthesis may play a significant role in the growth stimulation of tumor cells in soft tissue metastases as in primary tumors. TNF-alpha, IL-6 and COX-2, on the other hand, are unlikely to be implicated in this up-regulation.     

Intron variants of the p53 gene are associated with increased risk for ovarian cancer but not in carriers of BRCA1 or BRCA2 germline mutations.

Two biallelic polymorphisms in introns 3 and 6 of the p53 gene were analysed for a possible risk-modifying effect for ovarian cancer. Germline DNA was genotyped from 310 German Caucasian ovarian cancer patients and 364 healthy controls. We also typed 124 affected and 276 unaffected female carriers with known deleterious BRCA1 or BRCA2 germline mutation from high-risk breast-ovarian cancer families. Genotyping was based on PCR and high-resolution gel electrophoresis. German ovarian cancer patients who carried the rare allele of the MspI restriction fragment length polymorphism (RELP) in intron 6 were found to have an overall 1.93-fold increased risk (95% confidence internal (CI) 1.27-2.91) which further increased with the age at diagnosis of 41-60 years (odds ratio (OR) 2.71, 95% CI 1.10-6.71 for 41-50 and OR 2.44, 95% CI 1.12-5.28 for 51-60). The 16 bp duplication polymorphism in intron 3 was in a strong linkage to the MspI RFLP. In BRCA1 or BRCA2 mutation carriers, no difference in allele frequency was observed for carriers affected or unaffected with ovarian cancer. Our data suggest that intronic polymorphisms of the p53 gene modify the risk for ovarian cancer patients but not in carriers with BRCA1 or BRCA2 mutations.     

Significance of intraoperative radiation therapy and high cumulative radiation doses in retroperitoneal soft tissue sarcoma

IntroductionIn retroperitoneal soft tissue sarcoma (STS) local recurrence (LR) rates remain high despite more aggressive surgical approaches. Since wide resection margins cannot be achieved in all patients, application of intraoperative radiation therapy (IORT) has been frequently discussed. Still, the significance of IORT in multimodal treatment of retroperitoneal STS remains unclear.Material and methodsPatients undergoing resection of primary or recurrent retroperitoneal STS at the University of Heidelberg Department of General, Visceral and Transplantation Surgery were retrospectively analyzed. Univariate Kaplan-Meyer and multivariate Cox regression analyses were performed to identify predictors of LR-free survival and to investigate the impact of IORT and high cumulative radiation doses. Analyses with propensity-score matched subgroups for IORT and cumulative radiation dose were performed to control for selection bias. Subgroup analyses for patients with retroperitoneal liposarcoma were likewise performed.Results272 patients were identified. Recurrent tumors, histology of dedifferentiated liposarcoma or unclassified sarcoma and microscopically incomplete resection were associated with decreased LR-free survival. In liposarcoma, only recurrent and dedifferentiated tumors were confirmed as poor prognostic factors concerning LR. IORT and cumulative radiation doses exceeding 60 Gy did not influence LR rates (estimated 5-year LR-free survival: IORT: 39%, non-IORT: 46%; p = 0.79).ConclusionIn this retrospective evaluation, additional application of IORT does not significantly influence oncological outcome in retroperitoneal soft tissue sarcoma. Randomized trials are needed to clarify the benefit of IORT.