PIK3CA-mutations in breast cancer.

PURPOSE: Phosphatidylinositide-3-kinase (PI3K) regulates proliferation and apoptosis; somatic PIK3CA-mutations may activate these processes. Aim of this study was to determine the prevalence of PIK3CA-mutations in a cohort of early stage breast cancer patients and the association to the course of disease. PATIENTS AND METHODS: From an unselected cohort of 1270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT01592825) 1123 tumours were tested for the three PIK3CA hotspot-mutations H1047R, E545K, and E542K by qPCR. Primary objectives were the prevalence of somatic PIK3CA-mutations and their association to tumour characteristics. Secondary objective was the association of PIK3CA-mutations to recurrence-free interval (RFI) and overall survival. RESULTS: PIK3CA-mutation rate was 26.7% (300 of 1123). PIK3CA-mutations were significantly more frequent in steroid hormone-receptor (SHR)-positive HER2-negative (31.4%), and G1 and G2 tumours (32.8%). Overall, we did not observe a significant association of PIK3CA-mutations to RFI. In SHR-positive BCs with PIK3CA-mutations, a strong trend for impaired  RFI was observed (adjusted HR 1.64, 95% CI 0.958-2.807), whilst in SHR-negative BCs PIK3CA-mutations were insignificantly associated with improved RFI (adjusted HR 0.49; 95% CI 0.152-1.597). Of note, we observed a significantly detrimental prognostic impact of PIK3CA-mutations on RFI in SHR-positive, HER2-negative BCs if only aromatase inhibitors were administered as adjuvant therapy (adjusted HR 4.44, 95% CI 1.385-13.920), whilst no impact was observed in tamoxifen treated patients. CONCLUSION: This cohort study speficies the overall mutation rate of PIK3CA in early breast cancer. The impact of PIK3CA-mutations on RFI and OS was heterogeneous. Our results suggest that estrogen deprivation failes to be active in case of PIK3CA-mutation.

Mid-trimester preterm premature rupture of membranes (PPROM): etiology, diagnosis, classification, international recommendations of treatment options and outcome.

Mid-trimester preterm premature rupture of membranes (PPROM), defined as rupture of fetal membranes prior to 28 weeks of gestation, complicates approximately 0.4%-0.7% of all pregnancies. This condition is associated with a very high neonatal mortality rate as well as an increased risk of long- and short-term severe neonatal morbidity. The causes of the mid-trimester PPROM are multifactorial. Altered membrane morphology including marked swelling and disruption of the collagen network which is seen with PPROM can be triggered by bacterial products or/and pro-inflammatory cytokines. Activation of matrix metalloproteinases (MMP) have been implicated in the mechanism of PPROM. The propagation of bacteria is an important contributing factor not only in PPROM, but also in adverse neonatal and maternal outcomes after PPROM. Inflammatory mediators likely play a causative role in both disruption of fetal membrane integrity and activation of uterine contraction. The "classic PPROM" with oligo/an-hydramnion is associated with a short latency period and worse neonatal outcome compared to similar gestational aged neonates delivered without antecedent PPROM. The "high PPROM" syndrome is defined as a defect of the chorio-amniotic membranes, which is not located over the internal cervical os. It may be associated with either a normal or reduced amount of amniotic fluid. It may explain why sensitive biochemical tests such as the Amniosure (PAMG-1) or IGFBP-1/alpha fetoprotein test can have a positive result without other signs of overt ROM such as fluid leakage with Valsalva. The membrane defect following fetoscopy also fulfils the criteria for "high PPROM" syndrome. In some cases, the rupture of only one membrane - either the chorionic or amniotic membrane, resulting in "pre-PPROM" could precede "classic PPROM" or "high PPROM". The diagnosis of PPROM is classically established by identification of nitrazine positive, fern positive watery leakage from the cervical canal observed during in specula investigation. Other more recent diagnostic tests include the vaginal swab assay for placental alpha macroglobulin-1 test or AFP and IGFBP1. In some rare cases amniocentesis and infusion of indigo carmine has been used to confirm the diagnosis of PPROM. The management of the PPROM requires balancing the potential neonatal benefits from prolongation of the pregnancy with the risk of intra-amniotic infection and its consequences for the mother and infant. Close monitoring for signs of chorioamnionitis (e.g. body temperature, CTG, CRP, leucocytes, IL-6, procalcitonine, amniotic fluid examinations) is necessary to minimize the risk of neonatal and maternal complications. In addition to delayed delivery, broad spectrum antibiotics of penicillin or cephalosporin group and/or macrolide and corticosteroids have been show to improve neonatal outcome [reducing risk of chorioamnionitis (average risk ratio (RR)=0.66), neonatal infections (RR=0.67) and abnormal ultrasound scan of neonatal brain (RR=0.67)]. The positive effect of continuous amnioinfusion through the subcutaneously implanted perinatal port system with amniotic fluid like hypo-osmotic solution in "classic PPROM" less than 28/0 weeks' gestation shows promise but must be proved in future prospective randomized studies. Systemic antibiotics administration in "pre-PPROM" without infection and hospitalization are also of questionable benefit and needs to be further evaluated in well-designed randomized prospective studies to evaluate if it is associated with any neonatal benefit as well as the relationship to possible adverse effect of antibiotics on to fetal development and neurological outcome.

Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors.

BACKGROUND: Breast cancer tumors are known to be highly heterogeneous and differences in their metabolic phenotypes, especially at protein level, are less well-understood. Profiling of metabolism-related proteins harbors the potential to establish new patient stratification regimes and biomarkers promoting individualized therapy. In our study, we aimed to examine the relationship between metabolism-associated protein expression profiles and clinicopathological characteristics in a large cohort of breast cancer patients. METHODS: Breast cancer specimens from 801 consecutive patients, diagnosed between 2009 and 2011, were investigated using reverse phase protein arrays (RPPA). Patients were treated in accordance with national guidelines in five certified German breast centers. To obtain quantitative expression data, 37 antibodies detecting proteins relevant to cancer metabolism, were applied. Hierarchical cluster analysis and individual target characterization were performed. Clustering results and individual protein expression patterns were associated with clinical data. The Kaplan-Meier method was used to estimate survival functions. Univariate and multivariate Cox regression models were applied to assess the impact of protein expression and other clinicopathological features on survival. RESULTS: We identified three metabolic clusters of breast cancer, which do not reflect the receptor-defined subtypes, but are significantly correlated with overall survival (OS, p ≤ 0.03) and recurrence-free survival (RFS, p ≤ 0.01). Furthermore, univariate and multivariate analysis of individual protein expression profiles demonstrated the central role of serine hydroxymethyltransferase 2 (SHMT2) and amino acid transporter ASCT2 (SLC1A5) as independent prognostic factors in breast cancer patients. High SHMT2 protein expression was significantly correlated with poor OS (hazard ratio (HR) = 1.53, 95% confidence interval (CI) = 1.10-2.12, p ≤ 0.01) and RFS (HR = 1.54, 95% CI = 1.16-2.04, p ≤ 0.01). High protein expression of ASCT2 was significantly correlated with poor RFS (HR = 1.31, 95% CI = 1.01-1.71, p ≤ 0.05). CONCLUSIONS: Our data confirm the heterogeneity of breast tumors at a functional proteomic level and dissects the relationship between metabolism-related proteins, pathological features and patient survival. These observations highlight the importance of SHMT2 and ASCT2 as valuable individual prognostic markers and potential targets for personalized breast cancer therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01592825 . Registered on 3 May 2012.

Successful Closure of Small Iatrogenic Chorioamniotic Membranes Defect After Fetoscopy with Laser: A Case Report.

Background: Fetoscopic laser coagulation of the placental anastomoses is the standard treatment for twin-to-twin transfusion syndrome (TTTS). Despite certain improvements in fetoscopic technique, every fourth fetoscopic procedure is still complicated by preterm premature rupture of membranes, leading to ascending infection, fetal demise, and/or preterm delivery. re-TTTS occurs after fetoscopy in 2­14% of cases, impairing the outcome. Case: A 26-year-old woman underwent laser coagulation of placental anastomoses because of stage III TTTS at 21/6 weeks of gestation. A microinvasive fetoscopic technique with 1-mm optic was used. Three weeks later, during a second fetoscopy because of re-TTTS, a defect of the chorioamniotic membranes of about 3 mm2 in area was visualized. This was without any signs of wound healing. We decided to perform laser coagulation with Nd:YAG laser of 10­30 W energy, moving from the wound's edge to the center until complete closure of the defect could be achieved. The patient gave birth at 34/0 weeks to 2 healthy female infants weighing 2,013 g and 1,712 g. Microscopic evaluation of chorioamniotic membranes found dystrophic calcification within the treated membranes; this had been covered by amniotic epithelium. Conclusion: Small iatrogenic amniotic membrane defects could be successfully treated by laser technique.

Treatment of Preterm Premature Rupture of Membranes with Oligo-/Anhydramnion Colonized by Multiresistant Bacteria with Continuous Amnioinfusion and Antibiotic Administrations through a Subcutaneously Implanted Intrauterine Port System: A Case Report.

Bacterial infection is one of the main causes of preterm premature rupture of membranes (PPROM) leading to preterm delivery, pulmonary hypoplasia, sepsis and joint deformities. Expectant management, broad-spectrum antibiotics and antenatal corticosteroids are routinely used in this condition with very limited success to prevent bacteremia, chorioamnionitis, funisitis and intra-amniotic infection syndrome. Here, we report a case in which we attempted to treat PPROM at 26+3 weeks of gestation with anhydramnion colonized by multiresistant Klebsiella. A perinatal port system was implanted subcutaneously at 28+0 weeks of gestation, enabling long-term continuous lavage of the amniotic cavity with a hypotonic aqueous composition similar to human amniotic fluid combined with intra-amniotic antibiotic application. The patient gave birth to a preterm female infant at 31+1 weeks without any signs of infection. The girl was discharged with a weight of 2,730 g in very good condition. In the follow-up examinations at 5 months and 1 year of age, there was no apparent neurological disturbance, developmental delay or Klebsiella colonization.

Prognostic Value of Tumour-Infiltrating Lymphocytes in an Unselected Cohort of Breast Cancer Patients.

Tumour-infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. We examined 1166 breast cancer patients from a prospective, multicentre cohort (Prognostic Assessment in Routine Application (PiA), n = 1270, NCT01592825) following recommendations from the International TILs Working Group. TIL quantification was performed using predefined groups and as a continuous variable in 10% increments. The primary objective was the distribution of TILs in different breast cancer types. The second objective was the association with the recurrence-free interval (RFI) and overall survival (OS). Stromal infiltration with more than 60% TILs appeared in 2% of hormone receptor (HR)-positive and HER2-negative tumours, in 9.8% of HER2-positive tumours (any HR) and 19.4% of triple-negative breast cancers (TNBCs). Each 10% increment was associated with an improvement in the prognosis in HER2-positive samples (RFI, hazard ratio 0.773, 95% CI 0.587-1.017; OS, hazard ratio 0.700, 95% CI 0.523-0.937). When defining exploratory cut-offs for TILs, the use of a 30% threshold for the HR-positive and HER2-negative group, a 20% threshold for the HER2 group and a 60% threshold for the TNBC group appeared to be the most suitable. TILs bore prognostic value, especially in HER2-positive breast cancer. For clinical use, additional research on the components of immune infiltration might be reasonable.

Factors influencing the agreement on histopathological assessments of breast biopsies among pathologists.

AIMS: It has been recommended that the histopathology results of core biopsies of the breast are categorized according to the B-categorization scheme. We measured the interobserver variability of the B-categorization of core biopsies of the breast. METHODS AND RESULTS: Core biopsies were taken among 765 women at the University of Halle between 2006 and 2008. All histological slides were reviewed in a blinded fashion by two experienced breast pathologists. We calculated observed and chance-corrected agreements (kappa) and 95% confidence intervals (CI). The prevalence of B3-B5 biopsies was 41.6%. The observed and weighted kappa agreement of the five-level B-categorization scheme was 0.87 (95% CI: 0.84 -0.89) and 0.89 (95% CI: 0.89-0.91), respectively. The most frequent disagreement was between B2 and B3 (47 of 103 disagreements, 45.6%). Overall, 49.5% of all disagreements were clinically relevant disagreements that would imply different therapeutic strategies. Agreement was modified by referral group, Breast Imaging Reporting and Data System (BIRADS) level, radiological breast density, imaging guidance and application of immunohistological staining. CONCLUSIONS: Interobserver agreement of the B-categorization scheme was high and was modified by referral status, level of radiological suspicion of breast cancer, breast density, imaging guidance of core biopsies and requirement of additional immunohistological staining.

Identifying High-Risk Triple-Negative Breast Cancer Patients by Molecular Subtyping.

INTRODUCTION: Triple-negative breast cancer (TNBC) is considered the most aggressive type of breast cancer (BC) with limited options for therapy. TNBC is a heterogeneous disease and tumors have been classified into TNBC subtypes using gene expression profiling to distinguish basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor (LAR), and one nonclassifiable group (called unstable). OBJECTIVES: The aim of this study was to verify the clinical relevance of molecular subtyping of TNBCs to improve the individual indication of systemic therapy. PATIENTS AND METHODS: Molecular subtyping was performed in 124 (82%) of 152 TNBC tumors that were obtained from a prospective, multicenter cohort including 1,270 histopathologically confirmed invasive, nonmetastatic BCs (NCT01592825). Treatment was guideline-based. TNBC subtypes were correlated with recurrence-free interval (RFI) and overall survival (OS) after 5 years of observation. RESULTS: Using PAM50 analysis, 87% of the tumors were typed as basal with an inferior clinical outcome compared to patients with nonbasal tumors. Using the TNBCtype-6 classifier, we identified 23 (15%) of TNBCs as LAR subtype. After standard adjuvant or neoadjuvant chemotherapy, patients with LAR subtype showed the most events for 5-year RFI (66.7 vs. 80.6%) and the poorest probability of 5-year OS (60.0 vs. 84.4%) compared to patients with non-LAR disease (RFI: adjusted hazard ratio [aHR] = 1.87, 95% confidence interval [CI] 0.69-5.05, p = 0.211; OS: aHR = 2.74, 95% CI 1.06-7.10, p = 0.037). CONCLUSION: Molecular analysis and subtyping of TNBC may be relevant to identify patients with LAR subtype. These cancers seem to be less sensitive to conventional chemotherapy, and new treatment options, including androgen receptor-blocking agents and immune checkpoint inhibitors, have to be explored.

Specific allelic variants of SNPs in the MDM2 and MDMX genes are associated with earlier tumor onset and progression in Caucasian breast cancer patients.

BACKGROUND: Genetic factors play a substantial role in breast cancer etiology. Genes encoding proteins that have key functions in the DNA damage response, such as p53 and its inhibitors MDM2 and MDMX, are most likely candidates to harbor allelic variants that influence breast cancer susceptibility. The aim of our study was to comprehensively analyze the impact of SNPs in the TP53, MDM2, and MDMX genes in conjunction with TP53 mutational status regarding the onset and progression of breast cancer. METHODS: In specimen from 815 breast cancer patients, five SNPs within the selected genes were analyzed: TP53 - Arg72Pro (rs1042522), MDM2 - SNP285 (rs2279744), SNP309 (rs117039649); MDMX - SNP31826 (rs1563828), and SNP34091 (rs4245739). Classification of the tumors was evaluated by histomorphology. Subtyping according hormone receptor status, HER2-status and proliferation rate enabled provision of the clinico-pathological surrogate of intrinsic subtypes. RESULTS: The homozygous C-allele of MDM2 SNP285 was significantly associated with a younger age-at-diagnosis of 44.2 years, in contrast to G/G- and G/C-patients (62.4, 62.7 yrs., respectively; p = 0.0007; log-Rank-test). In contrast, there was no difference regarding the age-at-diagnosis for patients with the respective genotypes of MDM2 SNP309 (p = 0.799; log-Rank-test). In patients with estrogen receptor (ER)-positive and TP53-mutated tumors, however, the T/T-genotype of the MDM2 SNP309 was significantly associated with an earlier average age-at-diagnosis compared with T/G+G/G-patients (53.5 vs. 68.2 yrs; p = 0.002; log-Rank-test). In the triple-negative subgroup, the G/G-patients had an average age-at-diagnosis of 51 years compared with 63 years for SNP309T carriers (p = 0.004; log-Rank-test) indicating a susceptibility of the G/G genotype for the development of triple negative breast cancer. Patients with the A/A-genotype of MDMX SNP31826 with ER-negative tumors were diagnosed 11 years earlier compared with patients and ER-positive tumors (53.2 vs. 64.4 yrs; p = 0.025, log-Rank-test). Furthermore, in luminal B-like patients (HER2-independent) the C/C-genotype of MDMX SNP34091 was significantly correlated with a decreased event-free survival compared with the A/A-genotype (p < 0.001; log-Rank-test). CONCLUSIONS: We showed that SNPs in the MDM2 and MDMX genes affect at least in part the onset and progression of breast cancer dependent on the ER-status. Our findings provide further evidence for the distinct etiological pathways in ER-negative and ER-positive breast cancers.

MR-guided intervention in women at high hereditary risk of breast cancer due to both family and personal history of breast cancer.

The purpose of this study was to assess the value of magnetic resonance imaging (MRI)-guided intervention in women with a significant hereditary susceptibility to breast cancer due to both family and personal history of breast cancer (heterozygote risk >20%). Thirty women were referred for MR-guided intervention. MR examinations (1.0 T, T1-weighted 3D FLASH, 0.15 mmol Gd-DTPA/kg body weight, prone position) were performed using a system which allows vacuum-assisted breast biopsy or wire localization. Histologic findings in 41 procedures revealed six invasive carcinomas, eight ductal carcinomas in situ and two atypical ductal hyperplasias. Twenty-three benign histologic results were verified by an MR-guided intervention, retrospective correlation of imaging and histology and by subsequent follow-up. In two lesions the indication dropped as the enhancing lesion was no longer visible on the date of planned intervention. Absent enhancement was confirmed by short-term re-imaging of the non-compressed breast and by follow-up.

Preservation of histopathologic assessment by use of a special technique for tissue inactivation by radiofrequency.

Thermoinactivation is under investigation as a new method of cancer treatment. Prior histopathologic verification has been indispensable. Our experiments show that thermoinactivation is in principle possible without destroying histopathologic morphology, if the temperature stays within certain limits. It could thus become applicable already before diagnostic biopsy, which might be used to avoid any potential spread during biopsy and before definitive surgery.

Axillary lymph node metastases: a statistical analysis of various parameters in MRI with USPIO.

PURPOSE: To assess the value of plain vs. iron oxide-enhanced MRI vs. the combined study (plain + postcontrast) based on qualitative and quantitative parameters of three pulse sequences. MATERIALS AND METHODS: Data from two sites were acquired using the same technique; therefore, this data could be pooled. T1W-SE, T2W-FSE, and 3D-PSIF were used before and 24-36 hours after MRI with ultra small particles of iron oxide (USPIO) was performed. A total of 52 lymph nodes (LNs) in nine patients (25 benign, 27 malignant) were evaluated by two readers who were visually and quantitatively blinded to the histology. Combinations of the following diagnostic parameters were compared using logistic regression analysis: the short-axis diameter of the LN, the signal distribution of the LN on postcontrast agent MRI (homogeneous or heterogeneous), and qualitatively and quantitatively determined signal changes of the LN following administration of contrast agent in the three evaluated sequences. RESULTS: Using pre- and postcontrast data, the optimized accuracy based on the statistically most significant parameters (LN diameter > 6 mm, visual assessment of signal change on T2W-SE) was 87% (81% sensitivity, 92% specificity). Precontrast data alone yielded 75% accuracy (63% sensitivity, 86% specificity). Postcontrast data alone yielded 75% accuracy (56% sensitivity, 96% specificity). CONCLUSION: Based on our results, USPIO-MRI improved the diagnosis of metastatic axillary LNs compared with precontrast MRI alone. Both pre- and postcontrast studies are needed. T1W-SE and T2W-PSIF did not yield significant additional information. This study may help to further improve the technique of USPIO imaging.

MR imaging of the contralateral breast in patients after breast-conserving therapy.

A study was undertaken to assess the diagnostic accuracy of contrast-enhanced MR mammography (MRM) of the contralateral breast in patients treated by breast-conserving therapy previously. A total of 119 patients underwent 145 standardized dynamic MR studies (1 T, T1-weighted 3D FLASH, 0.2 mmol Gd-DTPA/kg body weight). We retrospectively evaluated the results of conventional methods and MRM. A total of 11 contralateral carcinomas were present (detection rate 9%). The interval between treatment of the first primary and identification of contralateral malignancy was 9-80 months (mean 33 months). The MRM allowed detection of four otherwise occult malignancies. One of 11 cancer was missed on MRM due to benign appearance of enhancement. Compared with conventional methods MRM improved sensitivity (91 vs 64%) and specificity (90 vs 84%), respectively. This study suggests that additional MRM of the contralateral breast increases the diagnostic accuracy not only by enhancing the detection of second cancers but also by reducing false-positive results.