Immunophenotyping analysis in invasive micropapillary carcinoma of the breast: Role of CD24 and CD44 isoforms expression

We analyzed immunohistochemically the expression of CD24 and spliced variants of CD44v5 and v9 in invasive micropapillary carcinoma (IMPC) of the breast that is a rather aggressive tumor characterized by alteration of cells adhesion molecules, early lymph node metastases and poor prognosis. We analyzed 31 high-grade IMPCs and compared their expression to 22 high grade (G3) invasive ductal carcinomas of the breast (IDCs). We found a higher expression of CD24 in high-grade IMPCs with a peculiar inverted apical localization, compared to IDCs, showing a strong cytoplasmic staining; normal breast tissue resulted completely negative. IMPCs showed reduced expression of CD44v5 and CD44v9 compared with IDCs, but without a statistical significant difference. This study demonstrated that IMPC represents a distinct entity of breast carcinoma with high expression of CD24 with a typical inverted apical membrane pattern and reduction of CD44 isoforms v5 and v9, compared to IDCs. These features could explain the high lymph-vascular invasion propensity and higher metastatic capability of these tumors and could be a useful tool for a future targeted therapy.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

Background Clinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes. Methods A tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival. Results CD4+ lymphocytes were more prevalent than FOXP3+ TILs whereas IL-17+ TILs were rare. Increased numbers of total CD4+ and FOXP3+ TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (p < 0.001) higher CD4+ and FOXP3+ lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (p < 0.001) associated with higher FOXP3+ TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4+ infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3+/CD4+ ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033). Conclusions Ductal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4+ and FOXP3+ TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3+/CD4+ TILs in ductal carcinoma appears to represent an independent favorable prognostic factor.

ENU-induced phenovariance in mice: inferences from 587 mutations

Background We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Heart rate variability does not discriminate between different levels of haemodynamic responsiveness during surgical anaesthesia

BACKGROUND: Hypnotic depth but not haemodynamic responsiveness is measured with EEG-based monitors. In this study we compared heart rate variability (HRV) in unstimulated patients and stimulation-induced HRV at different levels of anaesthesia. METHODS: A total of 95 ASA I or II patients were randomly assigned to five groups (Group 1: BIS 45(5), remifentanil 1 ng ml(-1); Group 2: BIS 45(5), remifentanil 2 ng ml(-1); Group 3: BIS 45(5), remifentanil 4 ng ml(-1); Group 4: BIS 30(5), remifentanil 2 ng ml(-1); Group 5: BIS 60(5), remifentanil 2 ng ml(-1)). A time- and frequency-domain analysis of the RR interval (RRI) from the electrocardiogram was performed. HRV before induction, before and after a 5 s tetanic stimulus of the ulnar nerve, and before and after tracheal intubation was compared between groups, between stimuli, and between responders to intubation [systolic arterial pressure (SAP) increase >20 mm Hg, a maximal heart rate (HR) after intubation >90 min(-1) or both] and non-responders (anova). RESULTS: Induction of anaesthesia significantly lowered HR and HRV. Mean RRI before stimulation was higher in G3 than in G1, G2, and G4 (P < 0.001), whereas the other HRV parameters were similar. Intubation induced a greater HRV response than tetanic stimulation. The mean RRI after intubation was lower in G3 compared with the other groups and the sd of the RRI after tetanic stimulation was lower in G3 compared with G5. Otherwise, unstimulated HRV and stimulation-induced HRV were similar in responders and non-responders. CONCLUSION: HRV parameters discriminate between awake and general anaesthesia, are different after tracheal intubation and a 5 s ulnar nerve stimulation, but do not discriminate between different levels of haemodynamic responsiveness during surgical anaesthesia.

A new standardized treadmill walking test requiring low motor skills in children aged 4-10 years

Exercise intolerance may be reported by parents of young children with respiratory diseases. There is, however, a lack of standardized exercise protocols which allow verification of these reports especially in younger children. Consequently the aims of this pilot study were to develop a standardized treadmill walking test for children aged 4-10 years demanding low sensorimotor skills and achieving high physical exhaustion. In a prospective experimental cross sectional pilot study, 33 healthy Caucasian children were separated into three groups: G1 (4-6 years, n = 10), G2 (7-8 years, n = 12), and G3 (9-10 years, n = 11). Children performed the treadmill walking test with increasing exercise levels up to peak condition with maximal exhaustion. Gas exchange, heart rate, and lactate were measured during the test, spirometry before and after. Parameters were statistically calculated at all exercise levels as well as at 2 and 4 mmol/L lactate level for group differences (Kruskal-Wallis H-test, alpha = 0.05; post hoc: Mann-Whitney U-test with Bonferroni correction alpha = 0.05/n) and test-retest differences (Wilcoxon-rank-sum test) with SPSS. The treadmill walking test could be demonstrated to be feasible with a good repeatability within groups for most of the parameters. All children achieved a high exhaustion level. At peak level under exhaustion condition only the absolute VO2 and VCO2 differed significantly between age groups. In conclusion this newly designed treadmill walking test indicates a good feasibility, safety, and repeatability. It suggests the potential usefulness of exercise capacity monitoring for children aged from early 4 to 10 years. Various applications and test modifications will be investigated in further studies.

Arctic patterned-ground ecosystems: A synthesis of field studies and models along a North American Arctic Transect

Arctic landscapes have visually striking patterns of small polygons, circles, and hummocks. The linkages between the geophysical and biological components of these systems and their responses to climate changes are not well understood. The "Biocomplexity of Patterned Ground Ecosystems" project examined patterned-ground features (PGFs) in all five Arctic bioclimate subzones along an 1800-km trans-Arctic temperature gradient in northern Alaska and northwestern Canada. This paper provides an overview of the transect to illustrate the trends in climate, PGFs, vegetation, n-factors, soils, active-layer depth, and frost heave along the climate gradient. We emphasize the thermal effects of the vegetation and snow on the heat and water fluxes within patterned-ground systems. Four new modeling approaches build on the theme that vegetation controls microscale soil temperature differences between the centers and margins of the PGFs, and these in turn drive the movement of water, affect the formation of aggradation ice, promote differential soil heave, and regulate a host of system propel-ties that affect the ability of plants to colonize the centers of these features. We conclude with an examination of the possible effects of a climate wan-ning on patterned-ground ecosystems.

The EmsB tandemly repeated multilocus microsatellite: a new tool to investigate genetic diversity of Echinococcus granulosus sensu lato

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People's Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE.

Accelerated partial breast irradiation with interstitial brachytherapy as second conservative treatment for ipsilateral breast tumour recurrence: multicentric study of the GEC-ESTRO Breast Cancer Working Group

PURPOSE To analyse the clinical outcome after salvage lumpectomy and multi-catheter brachytherapy (MCB) for ipsilateral breast tumour recurrence (IBTR). MATERIAL AND METHODS Between 09/00 and 09/10, 217 patients presenting an IBTR underwent lumpectomy and MCB (low, pulsed, or high-dose rate). Survival rates without second local recurrence (2nd LR), distant metastasis (DM), and overall survival (OS) were analysed as well as late effects and cosmetic results. Univariate and multivariate analyses (MVA) based on IBTR data were performed to find prognostic factors for 2nd LR, DM, and OS. RESULTS Median follow-up after the IBTR was 3.9 years [range: 1.1-10.3]. Five and 10-year actuarial 2nd LR rates were 5.6% [range: 1.5-9.5] and 7.2% [range: 2.1-12.1], respectively. Five and 10-year actuarial DM rates were 9.6% [range: 5.7-15.2] and 19.1% [range: 7.8-28.3], respectively. Five and 10-year actuarial OS rates were 88.7% [range: 83.1-94.8] and 76.4% [range: 66.9-87.3], respectively. In MVA, histological grade was prognostic factor for 2nd LR (p=0.008) and OS (p=0.02); while tumour size was prognostic factor for DM (p=0.03). G3-4 complication rate was 11%. Excellent/good cosmetic result was achieved in 85%. CONCLUSION This study suggests that in case of IBTR, lumpectomy plus MCB is feasible and effective in preventing 2nd LR with an OS rate at least equivalent to those achieved with salvage mastectomy.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Detection of pulmonary amylase activity in exhaled breath condensate

Amylase activity in exhaled breath condensate (EBC) is usually interpreted as an indication of oropharyngeal contamination despite the fact that amylase can be found in pulmonary excretions. The aim of this study was to recruit and refine an amylase assay in order to detect amylase activity in any EBC sample and to develop a method to identify EBC samples containing amylase of pulmonary origin. EBC was collected from 40 volunteers with an EcoScreen condenser. Amylase assays and methods to discriminate between oropharyngeal and pulmonary proteins were tested and developed using matched EBC and saliva samples. Our refined 2-chloro-4-nitrophenyl-α-D-maltotriosid (CNP-G3) assay was 40-fold more sensitive than the most sensitive commercial assay and allowed detection of amylase activity in 30 µl of EBC. We developed a dot-blot assay which allowed detection of salivary protein in saliva diluted up to 150 000-fold. By plotting amylase activity against staining intensity we identified a few EBC samples with high amylase activity which were aligned with diluted saliva. We believe that EBC samples aligned with diluted saliva contain amylase activity introduced during EBC collection and that all other EBC samples contain amylase activity of pulmonary origin and are basically free of oropharyngeal protein contamination.

A reliable risk score for stage IV esophagogastric cancer

BACKGROUND The role of surgery for patients with metastatic esophagogastric adenocarcinoma (EGC) is not defined. The purpose of this study was to define selection criteria for patients who may benefit from resection following systemic chemotherapy. METHODS From 1987 to 2007, 160 patients presenting with synchronous metastatic EGC (cT3/4 cNany cM0/1 finally pM1) were treated with chemotherapy followed by resection of the primary tumor and metastases. Clinical and histopathological data, site and number of metastases were analyzed. A prognostic score was established and validated in a second cohort from another academic center (n = 32). RESULTS The median survival (MS) in cohort 1 was 13.6 months. Significant prognostic factors were grading (p = 0.046), ypT- (p = 0.001), ypN- (p = 0.011) and R-category (p = 0.015), lymphangiosis (p = 0.021), clinical (p = 0.004) and histopathological response (p = 0.006), but not localization or number of metastases. The addition of grading (G1/2:0 points; G3/4:1 points), clinical response (responder: 0; nonresponder: 1) and R-category (complete:0; R1:1; R2:2) defines two groups of patients with significantly different survival (p = 0.001) [low risk group (Score 0/1), n = 22: MS 35.3 months, 3-year-survival 47.6%); high risk group (Score 2/3/4) n = 126: MS 12.0 months, 3-year-survival 14.2%]. The score showed a strong trend in the validation cohort (p = 0.063) [low risk group (MS not reached, 3-year-survival 57.1%); high risk group (MS 19.9 months, 3-year-survival 6.7%)]. CONCLUSION We observed long-term survival after resection of metastatic EGC. A simple clinical score may help to identify a subgroup of patients with a high chance of benefit from resection. However, the accurate estimation of achieving a complete resection, which is an integral element of the score, remains challenging.

Comparison of 68Ga-DOTANOC and 68Ga-DOTATATE PET/CT within patients with gastroenteropancreatic neuroendocrine tumors

Somatostatin receptor PET tracers such as [68Ga-DOTA,1-Nal3]-octreotide (68Ga-DOTANOC) and [68Ga-DOTA,Tyr3]-octreotate (68Ga-DOTATATE) have shown promising results in patients with neuroendocrine tumors, with a higher lesion detection rate than is achieved with 18F-fluorodihydroxyphenyl-l-alanine PET, somatostatin receptor SPECT, CT, or MR imaging. 68Ga-DOTANOC has high affinity for somatostatin receptor subtypes 2, 3, and 5 (sst2,3,5). It has a wider receptor binding profile than 68Ga-DOTATATE, which is sst2-selective. The wider receptor binding profile might be advantageous for imaging because neuroendocrine tumors express different subtypes of somatostatin receptors. The goal of this study was to prospectively compare 68Ga-DOTANOC and 68Ga-DOTATATE PET/CT in the same patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and to evaluate the clinical impact of 68Ga-DOTANOC PET/CT. Methods: Eighteen patients with biopsy-proven GEP-NETs were evaluated with 68Ga-DOTANOC and 68Ga-DOTATATE using a randomized crossover design. Labeling of DOTANOC and DOTATATE with 68Ga was standardized using a fully automated synthesis device. PET/CT findings were compared with 3-phase CT scans and in some patients with MR imaging, 18F-FDG PET/CT, and histology. Uptake in organs and tumor lesions was quantified and compared by calculation of maximum standardized uptake values (SUVmax) using volume computer-assisted reading. Results: Histology revealed low-grade GEP-NETs (G1) in 4 patients, intermediate grade (G2) in 7, and high grade (G3) in 7. 68Ga-DOTANOC and 68Ga-DOTATATE were false-negative in only 1 of 18 patients. In total, 248 lesions were confirmed by cross-sectional and PET imaging. The lesion-based sensitivity of 68Ga-DOTANOC PET was 93.5%, compared with 85.5% for 68Ga-DOTATATE PET (P = 0.005). The better performance of 68Ga-DOTANOC PET is attributed mainly to the significantly higher detection rate of liver metastases rather than tumor differentiation grade. Multivariate analysis revealed significantly higher SUVmax in G1 tumors than in G3 tumors (P = 0.009). This finding was less pronounced with 68Ga-DOTANOC (P > 0.001). Altogether, 68Ga-DOTANOC changed treatment in 3 of 18 patients (17%). Conclusion: The sst2,3,5-specific radiotracer 68Ga-DOTANOC detected significantly more lesions than the sst2-specific radiotracer 68Ga-DOTATATE in our patients with GEP-NETs. The clinical relevance of this finding has to be proven in larger studies.