Correction: SNiPER: a novel hypermethylation biomarker panel for liquid biopsy based early breast cancer detection.

[This corrects the article DOI: 10.18632/oncotarget.27303.].

SNiPER: a novel hypermethylation biomarker panel for liquid biopsy based early breast cancer detection.

INTRODUCTION: Mammography is the gold standard for early breast cancer detection, but shows important limitations. Blood-based approaches on basis of cell-free DNA (cfDNA) provide minimally invasive screening tools to characterize epigenetic alterations of tumor suppressor genes and could serve as a liquid biopsy, complementing mammography. METHODS: Potential biomarkers were identified from The Cancer Genome Atlas (TCGA), using HumanMethylation450-BeadChip data. Promoter methylation status was evaluated quantitatively by pyrosequencing in a serum test cohort (n = 103), a serum validation cohort (n = 368) and a plasma cohort (n = 125). RESULTS: SPAG6, NKX2-6 and PER1 were identified as novel biomarker candidates. ITIH5 was included on basis of our previous work. In the serum test cohort, a panel of SPAG6 and ITIH5 showed 63% sensitivity for DCIS and 51% sensitivity for early invasive tumor (pT1, pN0) detection at 80% specificity. The serum validation cohort revealed 50% sensitivity for DCIS detection on basis of NKX2-6 and ITIH5. Furthermore, an inverse correlation between methylation frequency and cfDNA concentration was uncovered. Therefore, markers were tested in a plasma cohort, achieving a 64% sensitivity for breast cancer detection using SPAG6, PER1 and ITIH5. CONCLUSIONS: Although liquid biopsy remains challenging, a combination of SPAG6, NKX2-6, ITIH5 and PER1 (SNiPER) provides a promising tool for blood-based breast cancer detection.

Fat necrosis and parenchymal scarring after breast-conserving surgery and radiotherapy with an intraoperative electron or fractionated, percutaneous boost: a retrospective comparison.

BACKGROUND: The aim of this retrospective analysis was to evaluate mammographic changes such as fat necroses and parenchymal scarring in the breast tissue within the first 3 years after breast-conserving surgery (BCS) and whole-breast irradiation with an additional intraoperative electron boost (IO-B) versus fractionated percutaneous boost (FP-B). METHODS: A total of 53 breast cancer patients (stage I/II) treated between 2006 and 2008 were included. All patients underwent BCS followed by fractionated whole-breast radiotherapy with a total dose and single dose of 50.4 and 1.8 Gy. Twenty patients had 10 Gy IO-B using electrons, and 33 patients were treated with a FP-B with 10.8 Gy. The IO-B was performed using the mobile linear accelerator NOVAC 7. The follow-up mammograms were focused on fat necroses, parenchymal scarring and skin thickening. RESULTS: Fat necroses occurred significantly more frequently in IO-B patients compared to FP-B patients (50.0 vs. 18.2 %). The fat necroses were mammographically detected a median of 17 versus 23 months post-surgery for the IO-B versus FP-B patients. The median size of fat necroses was 24 (14-30) mm for the IO-B group and 14 (4-53) mm for the FP-B group. Parenchymal scarring in the grade 3-4 tumor bed area was seen significantly more frequently in the IO-B patients (55.0 vs. 21.2 %). Skin thickening did not differ significantly. CONCLUSION: The IO-B led to significantly more fat necroses and local restricted parenchymal scarring in our analysis.

Mastectomy trends for early-stage breast cancer: a report from the EUSOMA multi-institutional European database.

INTRODUCTION: Recent single-institution reports have shown increased mastectomy rates during the last decade. Further studies aiming to determine if these reports could be reflecting a national trend in the United States of America (US) have shown conflicting results. We report these trends from a multi-institutional European database. PATIENTS AND METHODS: Our source of data was the eusomaDB, a central data warehouse of prospectively collected information of the European Society of Breast Cancer Specialists (EUSOMA). We identified patients with newly diagnosed unilateral early-stage breast cancer (stages 0, I or II) to examine rates and trends in surgical treatment. RESULTS: A total of 15,369 early-stage breast cancer cases underwent surgery in 13 Breast Units from 2003 to 2010. Breast conservation was successful in 11,263 cases (73.3%). Adjusted trend by year showed a statistically significant decrease in mastectomy rates from 2005 to 2010 (p = 0.003) with a progressive reduction of 4.24% per year. A multivariate model showed a statistically significant association of the following factors with mastectomy: age < 40 or ≥ 70 years, pTis, pT1mi, positive axillary nodes, lobular histology, tumour grade II and III, negative progesterone receptors and multiple lesions. CONCLUSION: Our study demonstrates that a high proportion of patients with newly diagnosed unilateral early-stage breast cancer from the eusomaDB underwent breast-conserving surgery. It also shows a significant trend of decreasing mastectomy rates from 2005 to 2010. Moreover, our study suggests mastectomy rates in the population from the eusomaDB are lower than those reported in the US.

Wnt signalling in human breast cancer: expression of the putative Wnt inhibitor Dickkopf-3 (DKK3) is frequently suppressed by promoter hypermethylation in mammary tumours.

INTRODUCTION: Expression of the putative Wnt signalling inhibitor Dickkopf-3 (DKK3) is frequently lost in human cancer tissues because of aberrant 5'-cytosine methylation within the DKK3 gene promoter. Since other Wnt signalling inhibitors have been reported to be targets of epigenetic inactivation in human breast cancer, we questioned if DKK3 expression is also epigenetically silenced during breast carcinogenesis and therefore might contribute to oncogenic Wnt signalling commonly found in this disease. METHODS: DKK3 mRNA expression and DKK3 promoter methylation were determined by RT-PCR, realtime PCR and methylation-specific PCR in breast cell lines (n = 9), normal breast tissues (n = 19) and primary breast carcinomas (n = 150), respectively. In vitro DNA demethylation was performed by incubating breast cell lines with 5-aza-2'-deoxycytidine and trichostatin A. DKK3 protein expression was analysed by immunohistochemistry in breast carcinomas (n = 16) and normal breast tissues (n = 8). Methylation data were statistically correlated with clinical patient characteristics. All statistical evaluations were performed with SPSS 14.0 software. RESULTS: DKK3 mRNA was downregulated in 71% (five of seven) of breast cancer cell lines and in 68% of primary breast carcinomas (27 of 40) compared with benign cell lines and normal breast tissues, respectively. A DNA demethylating treatment of breast cell lines resulted in strong induction of DKK3 mRNA expression. In tumourous breast tissues, DKK3 mRNA downregulation was significantly associated with DKK3 promoter methylation (p < 0.001). Of the breast carcinomas, 61% (92 of 150) revealed a methylated DKK3 promoter, whereas 39% (58 of 150) retained an unmethylated promoter. Loss of DKK3 expression in association with DKK3 promoter methylation (p = 0.001) was also confirmed at the protein level (p < 0.001). In bivariate analysis, DKK3 promoter methylation was not associated with investigated clinicopathological parameters except patient age (p = 0.007). CONCLUSIONS: DKK3 mRNA expression and consequently DKK3 protein expression become frequently downregulated during human breast cancer development due to aberrant methylation of the DKK3 promoter. Since DKK3 is thought to negatively regulate oncogenic Wnt signalling, DKK3 may be a potential tumour suppressor gene in normal breast tissue.

Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: a systematic expression analysis.

BACKGROUND: The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain - bikunin, encoded by AMBP - and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. METHODS: We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. RESULTS: We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. CONCLUSION: Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.

Epidemiology of Streptococcus agalactiae colonization in Germany.

Streptococcus agalactiae can cause severe pneumonia, sepsis and meningitis in neonates and remains one of the most prevalent causes of invasive neonatal infections. Maternal transmission of S. agalactiae during delivery can be prevented by prenatal screening and peripartal antibiotic prophylaxis. Implementation of CDC guidelines for group B streptococci (GBS) disease prevention resulted in a significant decline of invasive neonatal S. agalactiae infections in the USA. Similar national guidelines were issued in 2000 for Germany. However, the epidemiology of S. agalactiae colonization in Germany has not been investigated for more than 15 years and the impact these guidelines will have is therefore unknown. To assess colonization rates in Germany, we cultured vaginal and rectal swabs for S. agalactiae from pregnant and non-pregnant adult patients in the region of Aachen and Munich. Swabs were cultivated in selective broth medium for 24h and subsequently plated on blood agar plates according to the CDC recommendations. Colonies negative for catalase and pyrrolidonyl aminopeptidase were further differentiated by the CAMP test and a DNA probe specific for S. agalactiae. Rectal or vaginal colonization of S. agalactiae was found in 34 (16%) of 210 pregnant patients and in 41 (16%) of 250 non-pregnant women. S. agalactiae was found only in rectal swabs in 4% of pregnant and non-pregnant patients. For further characterization of the strains capsular serotypes and major surface protein antigens were determined by Ouchterlony immunodiffusion and PCR. Among the 75 different patient isolates serotype III was the most prevalent with 21 (28%) isolates, followed by 16 (21%) isolates of serotype II, 13 (17%) isolates of serotype Ia, 12 (16%) of serotype V, 11 (15%) of serotype Ib and only 2 (3%) isolates of serotype IV. The vast majority of all strains harbored genes for the major surface protein antigens, the alpha-C-protein or alpha-C-protein like antigens like Alp2-4, epsilon and Rib. These data show that S. agalactiae colonization is common in Germany and strict adherence to the guidelines for the preventions of GBS disease will result in peripartal antibiotic prophylaxis in up to 20% of all deliveries.

Systematic characterisation of GABRP expression in sporadic breast cancer and normal breast tissue.

The GABRP gene has been previously identified by in silico analysis of four million ESTs as a candidate gene differentially expressed in breast cancer. GABRP is located on chromosome 5q34 and it encodes the pi-subunit of the gamma-aminobutyric acid (GABA) receptor, a transmembrane protein expressed in the brain and several nonneuronal tissues. Using cDNA dot blot hybridisation (cancer profiling array), quantitative RT-PCR and non-radioisotopic in situ hybridisation (ISH), we have analysed GABRP expression in breast cancer and normal breast tissues as well as in nontumorigenic and tumorigenic breast cell lines. Analysis of the cancer profiling array revealed a more than 2-fold downregulation of GABRP (p < 0.001) in 76% of primary breast carcinomas (n = 50) compared to corresponding normal tissues. Quantitative RT-PCR in a panel of 23 normal human tissues showed that the GABRP expression level was most abundant in the normal breast tissues compared to other human tissues. GABRP downregulation in breast cancer was confirmed by quantitative RT-PCR in cryopreserved breast tumour and normal breast tissue specimens (n = 22), in archival formalin-fixed, paraffin-embedded tissue specimens (n = 32), as well as in breast cancer cell lines (n = 8). Furthermore, a significant downregulation of GABRP was noted in large (pT3-pT4) (p = 0.044) primary breast tumours. Non-radioisotopic ISH showed strong GABRP expression in normal epithelial and benign papilloma breast cells, but no signal could be detected in invasive ductal carcinoma. Altogether, these data suggest that GABRP is progressively down-regulated with tumour-progression, and that it may be useful as a prognostic marker in breast cancer.