Next Generation Sequencing of Reactive Stroma and Residual Breast Cancer Cells in Tumor Bed after Neoadjuvant Chemotherapy

Primary systemic or neoadjuvant chemotherapy of breast cancer has become a standard therapy option in locally advanced or predefined intrinsic subtypes such as triple negative or Her2 positive breast cancer. Neoadjuvant chemotherapy can result in complete pathological response without residual tumor cells (tumor bed) or partial response and non-response with different amounts of reactive stroma and residual tumor cells. The interaction between therapy regimens and tumoral driver mutations have been extensively studied, although the reactive stroma of the tumor bed received less attention. In this study, we characterized the mutational status of residual breast cancer cells and reactive tumor stroma devoid of residual tumor cells in partial or non-responders using next generation sequencing. Twenty-one post-therapeutic breast surgical specimens after neoadjuvant chemotherapy underwent pathogenic driver-mutation screening using microdissected residual breast cancer cells and in reactive stroma adjacent to tumor bed areas. In reactive stroma, no mutations could be validated. In residual breast cancer cells, mutations were detected in sixteen of twenty-one cases (76%). In nine of these twenty-one cases (43%), pathogenic driver mutations (PIK3CA, PTEN, TP53, FN1, PLAG1) were identified. Pathogenic driver-mutations are exclusively restricted to residual carcinoma cells and are absent in reactive stroma independently from intrinsic breast cancer subtypes or tumor stage. These data suggest that the absence of pathogenic mutations in a tumor bed without residual tumor cells may have prognostic implications after neoadjuvant chemotherapy

BMI1 is a target gene of E2F-1 and is strongly expressed in primary neuroblastomas

The oncogene BMI1 encodes a polycomb group transcription factor that is required for embryonic development and self-renewal of stem cells. Despite these important functions little is known about the regulation of BMI1 expression. A cDNA microarray based search for target genes of E2F-1 in neuroblastoma cells expressing a 4-OHT-regulated E2F-1-ER fusion protein identified many hitherto unknown E2F-1 regulated genes. A total of 10% of these genes, including BMI1, encode proteins that function primarily in the regulation of gene expression. The BMI1 promoter contains a putative E2F binding site that was required for the activation of a BMI1 promoter-dependent reporter construct by E2F-1. Chromatin immunoprecipitation revealed 4-OHT-dependent binding of E2F-1-ER and binding of endogenous E2F-1 to the BMI1 promoter in tumor cells. We have previously shown activation of the oncogene MYCN by E2F. Thus, in neuroblastomas deregulated E2F-1 can activate two oncogenes, MYCN and BMI1 that are known to co-operate in tumor formation. Consistent with a role of Bmi1 in neuroblastoma tumorigenesis we found strong Bmi1 expression in primary neuroblastomas. Our results reveal a novel link between E2F and polycomb transcription factors and suggest a role of Bmi1 in neuroblastomas

Enlarged scaling ranges for the KS-entropy and the information dimension

Numerical estimates of the Kolmogorov-Sinai entropy based on a finite amount of data decay towards zero in the relevant limits. Rewriting differences of block entropies as averages over decay rates, and ignoring all parts of the sample where these rates are uncomputable because of the lack of neighbours, yields improved entropy estimates. In the same way, the scaling range for estimates of the information dimension can be extended considerably. The improvement is demonstrated for experimental data.Comment: 5 pages, 6 figure

Loss of a FYN-regulated differentiation and growth arrest pathway in advanced stage neuroblastoma

AbstractTumor stage, age of patient, and amplification of MYCN predict disease outcome in neuroblastoma. To gain insight into the underlying molecular pathways, we have obtained expression profiles from 94 primary neuroblastoma specimens. Advanced tumor stages show a characteristic expression profile that includes downregulation of multiple genes involved in signal transduction through Fyn and the actin cytoskeleton. High expression of Fyn and high Fyn kinase activity are restricted to low-stage tumors. In culture, expression of active Fyn kinase induces differentiation and growth arrest of neuroblastoma cells. Expression of Fyn predicts long-term survival independently of MYCN amplification. Amplification of MYCN correlates with deregulation of a distinct set of genes, many of which are target genes of Myc. Our data demonstrate a causal role for Fyn kinase in the genesis of neuroblastoma

Epidemiology and Characteristics of Gastric Carcinoma in Childhood : An Analysis of Data from Population-Based and Clinical Cancer Registries

(1) Background: Gastric carcinoma is an exceptionally rare tumor in childhood. Little is known about the etiology, epidemiology, and clinical features of pediatric gastric carcinomas. This analysis aimed to fill this gap by increasing knowledge about the occurrence of gastric carcinoma in childhood. (2) Material and methods: Data from gastric carcinoma cases diagnosed between 2000 and 2017/2018 were retrieved from the Surveillance, Epidemiology, and End Results Program (SEER) and the German Center for Cancer Registry Data. Data from patients <20 years of age were analyzed for patient- and tumor-related characteristics. In addition, clinical data from patients with gastric carcinoma registered in the German Registry for Rare Pediatric Tumors (STEP) were analyzed for diagnostics, therapy, and outcome. (3) Results: Ninety-one cases of gastric carcinoma, mainly in adolescents, were identified in the epidemiologic cancer registries. Among patients with recorded staging data, advanced tumor stages were common (66.7%). Within the follow-up period covered, 63.7% of patients with clinical follow-up data died. Eight pediatric patients with gastric carcinoma were enrolled in the STEP registry, among whom two were patients with hereditary CDH1 mutations and another was a patient with Peutz–Jeghers syndrome. Three patients were found to have distinctly decreased immunoglobulin concentrations. All four patients in whom complete resection was achieved remained in remission. Three of the other four patients died despite multimodal therapy. (4) Conclusions: A combination of Helicobacter pylori infection and tumor predisposition and/or immunodeficiency appears to promote the development of gastric carcinoma in childhood. While patients with localized disease stages have a good chance of achieving durable remission through complete resection, patients with stage IV carcinomas face a dismal prognosis, highlighting the need to develop new strategies such as mutation-guided treatments

Impact of differing methodologies for serum miRNA-371a-3p assessment in stage I testicular germ cell cancer recurrence.

INTRODUCTION Current evidence shows that serum miR-371a-3p can identify disease recurrence in testicular germ cell tumour (TGCT) patients and correlates with tumour load. Despite convincing evidence showing the advantages of including miR-371a-3p testing to complement and overcome the classical serum tumour markers limitations, the successful introduction of a serum miRNA based test into clinical practice has been impeded by a lack of consensus regarding optimal methodologies and lack of a universal protocol and thresholds. Herein, we investigate two quantitative real-time PCR (qRT-PCR) based pipelines in detecting disease recurrence in stage I TGCT patients under active surveillance, and compare the sensitivity and specificity for each method. METHODS Sequential serum samples collected from 33 stage I TGCT patients undergoing active surveillance were analysed for miR-371a-3p via qRT-PCR with and without an amplification step included. RESULTS Using a pre-amplified protocol, all known recurrences were detected via elevated miR-371a-3p expression, while without pre-amplification, we failed to detect recurrence in 3/10 known recurrence patients. For pre-amplified analysis, sensitivity and specificity was 90% and 94.4% respectively. Without amplification, sensitivity dropped to 60%, but exhibited 100% specificity. DISCUSSION We conclude that incorporating pre-amplification increases sensitivity of miR-371a-3p detection, but produces more false positive results. The ideal protocol for quantification of miR-371a-3p still needs to be determined. TGCT patients undergoing active surveillance may benefit from serum miR-371a-3p quantification with earlier detection of recurrences compared to current standard methods. However, larger cross-institutional studies where samples are processed and data is analysed in a standardised manner are required prior to its routine clinical implementation

Impact of differing methodologies for serum miRNA-371a-3p assessment in stage I testicular germ cell cancer recurrence

INTRODUCTION Current evidence shows that serum miR-371a-3p can identify disease recurrence in testicular germ cell tumour (TGCT) patients and correlates with tumour load. Despite convincing evidence showing the advantages of including miR-371a-3p testing to complement and overcome the classical serum tumour markers limitations, the successful introduction of a serum miRNA based test into clinical practice has been impeded by a lack of consensus regarding optimal methodologies and lack of a universal protocol and thresholds. Herein, we investigate two quantitative real-time PCR (qRT-PCR) based pipelines in detecting disease recurrence in stage I TGCT patients under active surveillance, and compare the sensitivity and specificity for each method. METHODS Sequential serum samples collected from 33 stage I TGCT patients undergoing active surveillance were analysed for miR-371a-3p via qRT-PCR with and without an amplification step included. RESULTS Using a pre-amplified protocol, all known recurrences were detected via elevated miR-371a-3p expression, while without pre-amplification, we failed to detect recurrence in 3/10 known recurrence patients. For pre-amplified analysis, sensitivity and specificity was 90% and 94.4% respectively. Without amplification, sensitivity dropped to 60%, but exhibited 100% specificity. DISCUSSION We conclude that incorporating pre-amplification increases sensitivity of miR-371a-3p detection, but produces more false positive results. The ideal protocol for quantification of miR-371a-3p still needs to be determined. TGCT patients undergoing active surveillance may benefit from serum miR-371a-3p quantification with earlier detection of recurrences compared to current standard methods. However, larger cross-institutional studies where samples are processed and data is analysed in a standardised manner are required prior to its routine clinical implementation

Thyroid dysfunction and electrocardiographic changes in subjects without arrhythmias:a cross-sectional study of primary healthcare subjects from Copenhagen

ObjectiveThe objective of the present study was to investigate associations of both overt and subclinical thyroid dysfunction with common ECG parameters in a large primary healthcare population.DesignCross-sectional study.Setting and participantsThe study population comprised of primary healthcare patients in Copenhagen, Denmark, who had a thyroid function test and an ECG recorded within 7 days of each other between 2001 and 2011.Data sourcesThe Danish National Patient Registry was used to collect information regarding baseline characteristics and important comorbidities.Outcome measure and study groupsCommon ECG parameters were determined using Marquette 12SL software and were compared between the study groups. The study population was divided into five groups based on their thyroid status. Euthyroid subjects served as the reference group in all analyses.ResultsA total of 132 707 patients (age 52±17 years; 50% female) were included. Hyperthyroidism was significantly associated with higher heart rate and prolonged QTc interval with significant interaction with age (p&lt;0.009) and sex (p&lt;0.001). These associations were less pronounced for patients with higher age. Subclinical hyperthyroidism was associated with higher heart rate among females, and a similar trend was observed among males. Hypothyroidism was associated with slower heart rate and shorter QTc but only in women. Moreover, longer P-wave duration, longer PR interval and low voltage were observed in patients with both subclinical and overt hypothyroidism. However, the presence of low voltage was less pronounced with higher age (p=0.001).ConclusionBoth overt and subclinical thyroid disorders were associated with significant changes in important ECG parameters. Age and gender have significant impact on the association of thyroid dysfunction particularly on heart rate and QTc interval