A method for generating coherent spatially explicit maps of seasonal palaeoclimates from site-based reconstructions

We describe a method for reconstructing spatially explicit maps of seasonal palaeoclimate variables from site-based reconstructions. Using a 3D-Variational technique, the method finds the best statistically unbiased, and spatially continuous, estimate of the palaeoclimate anomalies through combining the site-based reconstructions and a prior estimate of the palaeoclimate state. By assuming a set of correlations in the error of the prior, the resulting climate is smoothed both from month to month and from grid cell to grid cell. The amount of smoothing can be controlled through the choice of two length-scale values. The method is applied to a set of reconstructions of the climate of the Last Glacial Maximum (ca. 21,000 years ago, yr BP) for southern Europe derived from pollen data with a prior derived from results from the third phase of the Palaeoclimate Intercomparison Project (PMIP3). We demonstrate how to choose suitable values for the smoothing length scales from the datasets used in the reconstruction

Clinical translation of [18F]ICMT-11 for measuring chemotherapy-induced caspase 3/7 activation in breast and lung cancer

Background: Effective anticancer therapy is thought to involve induction of tumour cell death through apoptosis and/or necrosis. [18F]ICMT-11, an isatin sulfonamide caspase-3/7-specific radiotracer, has been developed for PET imaging and shown to have favourable dosimetry, safety, and biodistribution. We report the translation of [18F]ICMT-11 PET to measure chemotherapy-induced caspase-3/7 activation in breast and lung cancer patients receiving first-line therapy. Results: Breast tumour SUVmax of [18F]ICMT-11 was low at baseline and unchanged following therapy. Measurement of M30/M60 cytokeratin-18 cleavage products showed that therapy was predominantly not apoptosis in nature. While increases in caspase-3 staining on breast histology were seen, post-treatment caspase-3 positivity values were only approximately 1%; this low level of caspase-3 could have limited sensitive detection by [18F]ICMT-11-PET. Fourteen out of 15 breast cancer patients responded to first–line chemotherapy (complete or partial response); one patient had stable disease. Four patients showed increases in regions of high tumour [18F]ICMT-11 intensity on voxel-wise analysis of tumour data (classed as PADS); response was not exclusive to patients with this phenotype. In patients with lung cancer, multi-parametric [18F]ICMT-11 PET and MRI (diffusion-weighted- and dynamic contrast enhanced-MRI) showed that PET changes were concordant with cell death in the absence of significant perfusion changes. Conclusion: This study highlights the potential use of [18F]ICMT-11 PET as a promising candidate for non-invasive imaging of caspase3/7 activation, and the difficulties encountered in assessing early-treatment responses. We summarize that tumour response could occur in the absence of predominant chemotherapy-induced caspase-3/7 activation measured non-invasively across entire tumour lesions in patients with breast and lung cancer

The effect of the stromal component of breast tumours on prediction of clinical outcome using gene expression microarray analysis

INTRODUCTION: The aim of this study was to examine the effect of the cellular composition of biopsies on the error rates of multigene predictors of response of breast tumours to neoadjuvant adriamycin and cyclophosphamide (AC) chemotherapy. MATERIALS AND METHODS: Core biopsies were taken from primary breast tumours of 43 patients prior to AC, and subsequent clinical response was recorded. Post-chemotherapy (day 21) samples were available for 16 of these samples. Frozen sections of each core were used to estimate the proportion of invasive cancer and other tissue components at three levels. Transcriptional profiling was performed using a cDNA array containing 4,600 elements. RESULTS: Twenty-three (53%) patients demonstrated a 'good' and 20 (47%) a 'poor' clinical response. The percentage invasive tumour in core biopsies collected from these patients varied markedly. Despite this, agglomerative clustering of sample expression profiles showed that almost all biopsies from the same tumour aggregated as nearest neighbours. SAM (significance analysis of microarrays) regression analysis identified 144 genes which distinguished high- and low-percentage invasive tumour biopsies at a false discovery rate of not more than 5%. The misclassification error of prediction of clinical response using microarray data from pre-treatment biopsies (on leave-one-out cross-validation) was 28%. When prediction was performed on subsets of samples which were more homogeneous in their proportions of malignant and stromal cells, the misclassification error was considerably lower (8%–13%, p < 0.05 on permutation). CONCLUSION: The non-tumour content of breast cancer samples has a significant effect on gene expression profiles. Consideration of this factor improves accuracy of response prediction by expression array profiling. Future gene expression array prediction studies should be planned taking this into account

Intratumoral Hydrogen Peroxide With Radiation Therapy in Locally Advanced Breast Cancer: Results From a Phase 1 Clinical Trial.

Purpose Hydrogen peroxide (H2O2) plays a vital role in normal cellular processes but at supraphysiological concentrations causes oxidative stress and cytotoxicity, a property that is potentially exploitable for the treatment of cancer in combination with radiation therapy (RT). We report the first phase 1 trial testing the safety and tolerability of intratumoral H2O2 + external beam RT as a novel combination in patients with breast cancer and exploratory plasma marker analyses investigating possible mechanisms of action.Methods and materials Twelve patients with breast tumors ≥3 cm (surgically or medically inoperable) received intratumoral H2O2 with either 36 Gy in 6 twice-weekly fractions (n = 6) or 49.5 Gy in 18 daily fractions (n = 6) to the whole breast ± locoregional lymph nodes in a single-center, nonrandomized study. H2O2 was mixed in 1% sodium hyaluronate gel (final H2O2 concentration 0.5%) before administration to slow drug release and minimize local discomfort. The mixture was injected intratumorally under ultrasound guidance twice weekly 1 hour before RT. The primary endpoint was patient-reported maximum intratumoral pain intensity before and 24 hours postinjection. Secondary endpoints included grade ≥3 skin toxicity and tumor response by ultrasound. Blood samples were collected before, during, and at the end of treatment for cell-death and immune marker analysis.Results Compliance with H2O2 and RT was 100%. Five of 12 patients reported moderate pain after injection (grade 2 Common Terminology Criteria for Adverse Events v4.02) with median duration 60 minutes (interquartile range, 20-120 minutes). Skin toxicity was comparable to RT alone, with maintained partial/complete tumor response relative to baseline in 11 of 12 patients at last follow-up (median 12 months). Blood marker analysis highlighted significant associations of TRAIL, IL-1β, IL-4, and MIP-1α with tumor response.Conclusions Intratumoral H2O2 with RT is well tolerated with no additional toxicity compared with RT alone. If efficacy is confirmed in a randomized phase 2 trial, the approach has potential as a cost-effective radiation response enhancer in multiple cancer types in which locoregional control after RT alone remains poor

Conditional independence relations among biological markers may improve clinical decision as in the case of triple negative breast cancers

The associations existing among different biomarkers are important in clinical settings because they contribute to the characterisation of specific pathways related to the natural history of the disease, genetic and environmental determinants. Despite the availability of binary/linear (or at least monotonic) correlation indices, the full exploitation of molecular information depends on the knowledge of direct/indirect conditional independence (and eventually causal) relationships among biomarkers, and with target variables in the population of interest. In other words, that depends on inferences which are performed on the joint multivariate distribution of markers and target variables. Graphical models, such as Bayesian Networks, are well suited to this purpose. Therefore, we reconsidered a previously published case study on classical biomarkers in breast cancer, namely estrogen receptor (ER), progesterone receptor (PR), a proliferative index (Ki67/MIB-1) and to protein HER2/neu (NEU) and p53, to infer conditional independence relations existing in the joint distribution by inferring (learning) the structure of graphs entailing those relations of independence. We also examined the conditional distribution of a special molecular phenotype, called triple-negative, in which ER, PR and NEU were absent. We confirmed that ER is a key marker and we found that it was able to define subpopulations of patients characterized by different conditional independence relations among biomarkers. We also found a preliminary evidence that, given a triple-negative profile, the distribution of p53 protein is mostly supported in 'zero' and 'high' states providing useful information in selecting patients that could benefit from an adjuvant anthracyclines/alkylating agent-based chemotherapy

Clinical response after two cycles compared to HER2, Ki-67, p53, and bcl-2 in independently predicting a pathological complete response after preoperative chemotherapy in patients with operable carcinoma of the breast

Introduction To investigate the predictive value of clinical and biological markers for a pathological complete remission after a preoperative dose-dense regimen of doxorubicin and docetaxel, with or without tamoxifen, in primary operable breast cancer. Methods Patients with a histologically confirmed diagnosis of previously untreated, operable, and measurable primary breast cancer (tumour (T), nodes (N) and metastases (M) score: T2-3(>= 3 cm) N0-2 M0) were treated in a prospectively randomised trial with four cycles of dose-dense (bi-weekly) doxorubicin and docetaxel (ddAT) chemotherapy, with or without tamoxifen, prior to surgery. Clinical and pathological parameters (menopausal status, clinical tumour size and nodal status, grade, and clinical response after two cycles) and a panel of biomarkers (oestrogen and progesterone receptors, Ki-67, human epidermal growth factor receptor 2 (HER2), p53, bcl-2, all detected by immunohistochemistry) were correlated with the detection of a pathological complete response (pCR). Results A pCR was observed in 9.7% in 248 patients randomised in the study and in 8.6% in the subset of 196 patients with available tumour tissue. Clinically negative axillary lymph nodes, poor tumour differentiation, negative oestrogen receptor status, negative progesterone receptor status, and loss of bcl-2 were significantly predictive for a pCR in a univariate logistic regression model, whereas in a multivariate analysis only the clinical nodal status and hormonal receptor status provided significantly independent information. Backward stepwise logistic regression revealed a response after two cycles, with hormone receptor status and lymph-node status as significant predictors. Patients with a low percentage of cells stained positive for Ki-67 showed a better response when treated with tamoxifen, whereas patients with a high percentage of Ki-67 positive cells did not have an additional benefit when treated with tamoxifen. Tumours overexpressing HER2 showed a similar response to that in HER2-negative patients when treated without tamoxifen, but when HER2-positive tumours were treated with tamoxifen, no pCR was observed. Conclusion Reliable prediction of a pathological complete response after preoperative chemotherapy is not possible with clinical and biological factors routinely determined before start of treatment. The response after two cycles of chemotherapy is a strong but dependent predictor. The only independent factor in this subset of patients was bcl-2. Trial registration number NCT0054382

In Silico Ascription of Gene Expression Differences to Tumor and Stromal Cells in a Model to Study Impact on Breast Cancer Outcome

Breast tumors consist of several different tissue components. Despite the heterogeneity, most gene expression analyses have traditionally been performed without prior microdissection of the tissue sample. Thus, the gene expression profiles obtained reflect the mRNA contribution from the various tissue components. We utilized histopathological estimations of area fractions of tumor and stromal tissue components in 198 fresh-frozen breast tumor tissue samples for a cell type-associated gene expression analysis associated with distant metastasis. Sets of differentially expressed gene-probes were identified in tumors from patients who developed distant metastasis compared with those who did not, by weighing the contribution from each tumor with the relative content of stromal and tumor epithelial cells in their individual tumor specimen. The analyses were performed under various assumptions of mRNA transcription level from tumor epithelial cells compared with stromal cells. A set of 30 differentially expressed gene-probes was ascribed solely to carcinoma cells. Furthermore, two sets of 38 and five differentially expressed gene-probes were mostly associated to tumor epithelial and stromal cells, respectively. Finally, a set of 26 differentially expressed gene-probes was identified independently of cell type focus. The differentially expressed genes were validated in independent gene expression data from a set of laser capture microdissected invasive ductal carcinomas. We present a method for identifying and ascribing differentially expressed genes to tumor epithelial and/or stromal cells, by utilizing pathologic information and weighted t-statistics. Although a transcriptional contribution from the stromal cell fraction is detectable in microarray experiments performed on bulk tumor, the gene expression differences between the distant metastasis and no distant metastasis group were mostly ascribed to the tumor epithelial cells of the primary breast tumors. However, the gene PIP5K2A was found significantly elevated in stroma cells in distant metastasis group, compared to stroma in no distant metastasis group. These findings were confirmed in gene expression data from the representative compartments from microdissected breast tissue. The method described was also found to be robust to different histopathological procedures

Eye bank issues: II. Preservation techniques: warm versus cold storage

Most of the tissue used for penetrating keratoplasty is issued through eye banks that store the corneoscleral button either in hypothermic storage at 2–6°C or in organ culture at 31–37°C

Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy