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Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that is associated with significantly high mortality. In spite of advances in IBC diagnoses, the prognosis is still poor compared to non-IBC. Due to the aggressive nature of the disease, we hypothesize that elevated levels of inflammatory mediators may drive tumorigenesis and metastasis in IBC patients. Utilizing IBC cell models and patient tumor samples, we can detect elevated NF-κB activity and hyperactivation of non-canonical drivers of NF-κB (nuclear factor kappaB)-directed inflammation such as tyrosine phosphorylated receptor-interacting protein kinase 2 (pY RIPK2), when compared to non-IBC cells or patients. Interestingly, elevated RIPK2 activity levels were present in a majority of pre-chemotherapy samples from IBC patients at the time of diagnosis to suggest that patients at diagnosis had molecular activation of NF-κB via RIPK2, a phenomenon we define as "molecular inflammation". Surprisingly, chemotherapy did cause a significant increase in RIPK2 activity and thus molecular inflammation suggesting that chemotherapy does not resolve the molecular activation of NF-κB via RIPK2. This would impact on the metastatic potential of IBC cells. Indeed, we can demonstrate that RIPK2 activity correlated with advanced tumor, metastasis, and group stage as well as body mass index (BMI) to indicate that RIPK2 might be a useful prognostic marker for IBC and advanced stage breast cancer.

We have previously identified a novel phenotypic dichotomy in breast cancer (BC) based on the response to a SRR2 (Sox2 regulatory region 2) reporter, with reporter responsive (RR) cells being more tumorigenic/stem-like than reporter unresponsive (RU) cells. Since the expression level of Sox2 is comparable between the two cell subsets, we hypothesized that post-translational modifications of Sox2 contribute to their differential reporter response and phenotypic differences. By liquid chromatography-mass spectrometry, we found Sox2 to be phosphorylated in RR but not RU cells. Threonine 116 is an important phosphorylation site, since transfection of the T116A mutant into RR cells significantly decreased the SRR2 reporter luciferase activity and the RR-associated phenotype. Oxidative stress-induced conversion of RU into RR cells was accompanied by Sox2 phosphorylation at T116 and increased Sox2-DNA binding. In a cohort of BC, we found significant correlations between the proportion of tumor cells immuno-reactive with anti-phosphorylated Sox2 T116 and a high tumor grade ( p = 0.006), vascular invasion ( p = 0.001) and estrogen receptor expression ( p = 0.032). In conclusion, our data suggests that phosphorylation of Sox2 T116 contributes to the tumorigenic/stem-like features in RR cells. Detection of phospho-Sox2 T116 may be useful in identifying a small subset of tumor cells carrying stem-like/tumorigenic features in BC., Competing Interests: The authors declare no conflicts of interest.

Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson's r = 0.909) and between users (Pearson's r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer.

A new automated MYC IHC classifier based on bivariate logistic regression is presented. The predictor relies on image analysis developed with the open-source ImageJ platform. From a histologic section immunostained for MYC protein, 2 dimensionless quantitative variables are extracted: (a) relative distance between nuclei positive for MYC IHC based on euclidean minimum spanning tree graph and (b) coefficient of variation of the MYC IHC stain intensity among MYC IHC-positive nuclei. Distance between positive nuclei is suggested to inversely correlate MYC gene rearrangement status, whereas coefficient of variation is suggested to inversely correlate physiological regulation of MYC protein expression. The bivariate classifier was compared with 2 other MYC IHC classifiers (based on percentage of MYC IHC positive nuclei), all tested on 113 lymphomas including mostly diffuse large B-cell lymphomas with known MYC fluorescent in situ hybridization (FISH) status. The bivariate classifier strongly outperformed the "percentage of MYC IHC-positive nuclei" methods to predict MYC+ FISH status with 100% sensitivity (95% confidence interval, 94-100) associated with 80% specificity. The test is rapidly performed and might at a minimum provide primary IHC screening for MYC gene rearrangement status in diffuse large B-cell lymphomas. Furthermore, as this bivariate classifier actually predicts "permanent overexpressed MYC protein status," it might identify nontranslocation-related chromosomal anomalies missed by FISH.

There is a pressing need for an objective decision tool to guide therapy for breast cancer patients that are estrogen receptor positive and HER2/neu negative. This subset of patients contains a mixture of luminal A and B tumors with good and bad outcomes, respectively. The 2 main current tools are on the basis of immunohistochemistry (IHC) or gene expression, both of which rely on the expression of distinct molecular groups that reflect hormone receptors, HER2/neu status, and most importantly, proliferation. Despite the success of a proprietary molecular test, definitive superiority of any method has not yet been demonstrated. Ki67 IHC scoring assessments have been shown to be poorly reproducible, whereas molecular testing is costly with a longer turnaround time. This work proposes an objective Ki67 index using image analysis that addresses the existing methodological issues of Ki67 quantitation using IHC on paraffin-embedded tissue. Intrinsic bias related to numerical assessment performed on IHC is discussed as well as the sampling issue related to the "peel effect" of tiny objects within a thin section. A new nonbiased stereological parameter (VV) based on the Cavalieri method is suggested for use on a double-stained Ki67/cytokeratin IHC slide. The assessment is performed with open-source ImageJ software with interobserver concordance between 3 pathologists being high at 93.5%. Furthermore, VV was found to be a superior method to predict an outcome in a small subset of breast cancer patients when compared with other image analysis methods being used to determine the Ki67 labeling index. Calibration methodology is also discussed to further this IHC approach.

We have previously identified a novel intra-tumoral dichotomy in triple-negative breast cancer (TNBC) based on the differential responsiveness to a reporter containing the Sox2 regulatory region-2 (SRR2), with reporter responsive (RR) cells being more stem-like than reporter unresponsive (RU) cells. Using bioinformatics, we profiled the protein-DNA binding motifs of SRR2 and identified Myc as one of the potential transcription factors driving SRR2 activity. In support of its role, Myc was found to be highly expressed in RR cells as compared to RU cells. Enforced expression of MYC in RU cells resulted in a significant increase in SRR2 activity, Myc-DNA binding, proportion of cellsexpressing CD44+/CD24-, chemoresistance and mammosphere formation. Knockdown of Myc using siRNA in RR cells led to the opposite effects. We also found evidence that the relatively high ERK activation in RR cells contributes to their high expression of Myc and stem-like features. Using confocal microscopy and patient samples, we found a co-localization between Myc and CD44 in the same cell population. Lastly, a high proportion of Myc-positive cells in tumors significantly correlated with a short patient survival. In conclusion, inhibition of the MAPK/ERK/Myc axis may be an effective approach in eliminating stem-like cells in TNBC.

We investigate the association between phosphorylated histone H3 (PhH3) and Oncotype DX recurrence score (RS). All invasive breast carcinoma with RS results from our city between 2007 and 2010 (n=47) were reviewed. Whole-tumor sections were stained for PhH3. Mitotic and PhH3 counts were performed and clinical charts reviewed. PhH3 correlated well with RS (r=0.69, P<0.001). Other correlations were: PhH3 versus mitotic count (r=0.87, P<0.001), PhH3 versus mitotic score (r=0.71, P<0.001), PhH3 versus modified Bloom-Richardson-Elston (MBR) grade (r=0.65, P<0.001), RS versus mitotic count (r=0.62, P<0.001), RS versus mitotic score (r=0.44, P=0.002), and RS versus MBR grade (r=0.49, P=0.001). Significant correlation between PhH3 and RS remained after controlling for mitotic count (r=0.39, P=0.007), mitotic score (r=0.60, P<0.001), MBR grade (r=0.56, P<0.001), and all 3 (r=0.37, P=0.014) by partial correlation. Two patients died of metastasis at 12 and 38 months after diagnosis. One had intermediate RS, and 1 high RS; both were in the top-third of PhH3 count. All other patients are alive and recurrence free. Correlation between PhH3 and RS was statistically significant in our cohort, and remained significant after controlling for traditional measures of proliferation. Given that RS has an established strong relationship with prognosis and therapy responsiveness, PhH3 may thus also be an important prognostic/predictive marker in breast cancer., Competing Interests: The authors declare no conflict of interest.

We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expression of 3 signature genes of RR cells (CD133, GPR49 and MUC15). Lastly, the H2O2-induced RU/RR conversion was detectable in a SCID mouse xenograft model and primary tumor cells. To conclude, the H2O2-induced RU/RR conversion has provided a novel model to study the acquisition of cancer stemness and plasticity.

Introduction: Aberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets., Methods: We used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples., Results: We found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples., Conclusions: In conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.

Introduction: Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals., Methods: Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays., Results: Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH., Conclusions: Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Mitotic count, PhH3, and MIB-1 are used as measures of the proportion of proliferating malignant cells in surgical pathology. They highlight different stages of the cell cycle, but little is known about how this affects their counts. This study assesses the strength of their correlations and attempts to determine the relationship between them. Proliferation counts for forty-nine consecutive cases of invasive breast carcinomas were analyzed, with the same tumor area on each stain counted using digital image analysis. The integrated optical density (IOD) of nuclei was measured as an approximation of nuclear DNA content. PhH3 strongly correlated with mitotic count (r = 0.94). Weaker correlations were found between MIB-1 versus PhH3 (r = 0.79) and mitotic count (r = 0.83). Nuclear IOD showed stronger correlation with MIB-1 (r = 0.37) than to mitotic count (r = 0.23) and PhH3 (r = 0.34). With evidence from a literature review, it is suggested that the weaker correlations with MIB-1 are not explained by count imprecision or error, but relies on temporal decorrelation between cell cycle phases. Consequences on correlation between these proliferative markers are illustrated by mathematical models.

Background: Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood., Methods: In-vitro invasion assay was used to evaluate whether the expression of Sox2 is linked to the invasiveness of MCF7 and ZR751 cells. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and/or Western blots were used to assess if Sox2 modulates the expression of factors known to regulate epithelial mesenchymal transition (EMT), such as Twist1. Chromatin immunoprecipitation (ChIP) was used to assess the binding of Sox2 to the promoter region of Twist1., Results: We found that siRNA knockdown of Sox2 expression significantly increased the invasiveness of MCF7 and ZR751 cells. However, when MCF7 cells were separated into two distinct subsets based on their differential responsiveness to the Sox2 reporter, the Sox2-mediated effects on invasiveness was observed only in 'reporter un-responsive' cells (RU cells) but not 'reporter responsive' cells (RR cells). Correlating with these findings, siRNA knockdown of Sox2 in RU cells, but not RR cells, dramatically increased the expression of Twist1. Accordingly, using ChIP, we found evidence that Sox2 binds to the promoter region of Twist1 in RU cells only. Lastly, siRNA knockdown of Twist1 largely abrogated the regulatory effect of Sox2 on the invasiveness in RU cells, suggesting that the observed Sox2-mediated effects are Twist1-dependent., Conclusion: Sox2 regulates the invasiveness of BC cells via a mechanism that is dependent on Twist1 and the transcriptional status of Sox2. Our results have further highlighted a new level of biological complexity and heterogeneity of BC cells that may carry significant clinical implications.

Objective: Color deconvolution relies on determination of unitary optical density vectors (OD(3D)) derived from pure constituent stains initially defined as intensity vectors in RGB space. OD(3D) can be defined in polar coordinates (phi, theta, radius); always being equal to one, radius can be ignored. Easier handling of unitary optical density 2D vectors (OD(2D)) is shown., Study Design: OD(2D) pure stains used in anatomical pathology were assessed as centroid values (phi, theta) with a measure of variance: inertia based on arc lengths between centroid value and sampled points. These variables were plotted on a stereographic projection plane. In order to assess pure stains OD(2D), different methods of sampling RGB pixels were tested and compared: (2) direct sampling of nuclei from preparations using (a) composite H&E and (b) hematoxylin only and (2) for any pure stain RGB image, different associated 8-bit masks (saturation, brightness and RGB average) were used for sampling and compared. Behaviors of phi, theta and inertia were obtained by moving threshold in 8-bit mask histograms. Phi and theta stability were tested against variable light intensity during image acquisition and by using 2 different image acquisition systems., Results: The more saturated RGB pixels are, the more stable phi, theta and inertia values are obtained. Different commercial hematoxylins have distinct OD(2D) characteristics. UltraView DAB stain shows high inertia and is angularly closer to usual counterstains than ultraView Red stain, which also has a lower inertia. Superior accuracy is expected from the latter stain. Phi and theta OD(2D) values are sensitive to light intensity variation, to the used imaging system and to the used objectives. An ImageJ plugin was designed to plot and interactively modify OD(2D) values with instant update of color deconvolution allowing heuristic segmentation., Conclusion: Utilization of polar OD(2D) eases statistical characterization of OD(3D) vectors: conditions of optimal sampling were demonstrated and various factors influencing OD(2D) stability were explored. Stereographic projection plane allows intuitive visualization of OD(3D) vectors as well as heuristic vectorial modification. All findings are not restricted to anatomical pathology but can be applied to bright field microscopy and subtractive color applications in general.

Germ cell tumor with somatic malignant transformation is an uncommon phenomenon occurring about 7% of all mediastinal teratomas. Among all transformed component, sarcoma appears to be the most frequent histology, followed by primitive neuroectodermal tumor (PNET) and adenocarcinoma. To our knowledge, there were 3 cases of colonic-type adenocarcinoma arising in a primary teratoma have been reported to date. However, none of them received chemotherapy directed to transformed histology given localized disease at presentation. We, therefore, report here the first case of patient who achieved good response from chemotherapy directed to transformed histology, which confirms the importance of chemotherapy regimen used.

Aims: Pan-cytokeratin (pan-CK) and low molecular weight cytokeratin (LMWCK) tests are the most common immunohistochemistry (IHC) tests used to support evidence of epithelial differentiation. Canadian Immunohistochemistry Quality Control (CIQC), a new provider of proficiency testing for Canadian clinical IHC laboratories, has evaluated the performance of Canadian IHC laboratories in two proficiency testing challenges for both pan-CK and LMWCK., Methods: CIQC has designed a 70-sample tissue microarray (TMA) for challenge 1 and a 30-sample TMA for challenge 2. There were 13 participants in challenge 1, and 62 in challenge 2. All results were evaluated and scored by CIQC assessors and compared with reference laboratory results., Results: Participating laboratories often produced false-negative results that ranged from 20% to 80%. False-positive results were also detected. About half of participating clinical laboratories have inappropriately calibrated IHC tests for pan-CK and LMWCK, which are the most commonly used markers for demonstration of epithelial differentiation. The great majority of laboratories were not aware of the problem with calibration of pan-CK and LMWCK tests because of inappropriate selection of external positive controls and samples for optimisation of these tests. Benign liver and kidney are the most important tissues to include as positive controls for both pan-CK and LMWCK., Conclusions: Participation in external quality assurance is important for peer comparison and proper calibration of IHC tests, which is also helpful for appropriate selection of positive control material and material for optimisation of the tests.

Background: Ultrasound (US) has an ever increasing scope in the evaluation of trauma, but relies greatly on operator experience. NASA has refined telesongraphy (TS) protocols for traumatic injury, especially in reference to mentoring inexperienced users. We hypothesized that such TS might benefit remote terrestrial caregivers. We thus explored using real-time US and video communication between a remote (Banff) and central (Calgary) site during acute trauma resuscitations., Methods: A existing internet link, allowing bidirectional videoconferencing and unidirectional US transmission was used between the Banff and Calgary ERs. Protocols to direct or observe an extended focused assessment with sonography for trauma (EFAST) were adapted from NASA algorithms. A call rota was established. Technical feasibility was ascertained through review of completed checklists. Involved personnel were interviewed with a semistructured interview., Results: In addition to three normal volunteers, 20 acute clinical examinations were completed. Technical challenges requiring solution included initiating US; audio and video communications; image freezing; and US transmission delays. FAST exams were completed in all cases and EFASTs in 14. The critical anatomic features of a diagnostic examination were identified in 98% of all FAST exams and a 100% of all EFASTs that were attempted. Enhancement of clinical care included confirmation of five cases of hemoperitoneum and two pneumothoraces (PTXs), as well as educational benefits. Remote personnel were appreciative of the remote direction particularly when instructions were given sequentially in simple, nontechnical language., Conclusions: The remote real-time guidance or observation of an EFAST using TS appears feasible. Most technical problems were quickly overcome. Further evaluation of this approach and technology is warranted in more remote settings with less experienced personnel.

The determination of grade and stage in renal cell carcinomas (RCCs) often fails to predict the actual clinical outcome for individual patients. The aim of the present work was to investigate whether it is possible to significantly improve the prognostic accuracy of the grading system by using the combination of two independent computer-assisted microscopy techniques. The first technique relates to the quantitative description of morphonuclear and nuclear DNA content features by means of the image analysis of Feulgen-stained cell nuclei, and the second quantitatively characterizes tumor growth by means of different cell kinetic parameters. These parameters consist of a duplication of a time-related parameter determined by means of the technique of using silver-stained proteins in interphase nucleolar organizer regions (AgNOR), a proliferation index determined by means of MIB-1 immunohistochemistry, and an apoptotic index determined by means of the terminal dUTP nick end labeling technique. The prognostic value of these quantitative features was investigated in a series of 60 RCCs. The quantitative analysis of Feulgen-stained nuclei made it possible to identify subgroups of patients with significantly different prognoses in both grade II and grade III RCCs. We labeled the RCCs associated with the most favorable prognoses as grade II- and III- and those with the least favorable ones as grade II+ and III+. The two most important kinetic variables to identify patients with different clinical outcomes were the MIB-1 index and the mean AgNOR area in the MIB-1-positive cells. Three significantly different survival curves were obtained for the 53 grade II and III RCC patients. Our results show that conventional RCC grading can be significantly improved by the quantitative analysis of Feulgen-stained nuclei, by cell kinetic parameter determination, and, more importantly, by combining the proliferation index with the mean AgNOR area parameter., (Copyright 2000 Wiley-Liss, Inc.)

This study investigated the actual growth rate of 30 low stage operable non-small cell lung carcinomas, including disease-free surviving and deceased patients. The actual growth rate was defined as the cell production rate and was calculated from the growth fraction and the cell cycle time of each tumor at the time of surgical resection. The growth fraction was assessed by the Ki67 index while the cell cycle time was assumed to be reflected by the AgNORs content in the cells positive for Ki67. AgNORs content was evaluated by means of image analysis of double-stained AgNOR-Ki67 tissue section. The actual growth rate did not discriminate between the disease-free surviving and deceased patients but the AgNORs content in Ki67 cells correlated with the survival time of those patients who died of the tumor. Patients expressing a small AgNORs content, which might indicate a long cell cycle, may die but later; patients with a high AgNORs content, which might indicate a short cell cycle, die early or will survive. A twilight curve was derived from this data and might provide new prognostic indicators.

This paper reports on cellular sociology, which consists of modeling tissular architecture based on graph theory. Voronoi's diagram was chosen to build the models. This diagram derives from a cell neighborhood concept and generates parameters which objectively represent tissue architecture. Minimal spanning tree (MST) is probably the more frequently used among graphs and successfully discriminates different grades of pathological process. However, Voronoi's diagram is more comprehensive and a more complete representation of architecture with the advantage of stability. The lung neuroendocrine tumor classification is far from being consensual, especially for lesions which don't fall in with typical carcinoid and small cell carcinoma groups. By comparing architectural models of 20 neuroendocrine tumors of the lung, this work supports the morphologic spectrum concept of these tumors and also supports the recently proposed concept of large-cell neuroendocrine tumors of the lung. Finally, architectural parameters separate small-cell-lung carcinomas from neuroendocrine non-small-cell lung carcinomas.

This paper reports on the phenomenon of heterogeneity of DNA distribution in colorectal carcinoma. The aim of this study was to find a new strategy in sampling methodology as a solution to the heterogeneity problem by studying ploidy topography. The study was carried out by using image analysis for densitometric measurements of tissue imprints of 19 colorectal specimens after Feulgen staining. Heterogeneity of ploidy in colorectal carcinoma is well known; therefore, we wished to determine whether this heterogeneity is random within different parts of a colorectal tumour. For each tumour, five systematic and reproducible samples were taken from the peripheral, intermediate and central tumoural areas. In addition two samples were taken from adjacent non-tumoural areas: one from the proximal and the other from the distal site with respect to the neoplastic lesion. By using image analysis three parameters were obtained. Mean DNA content was computed for each sample and expressed in arbitrary units (DNA-a.u.) from the measurement of integrated optical density according to the Beer Lambert law. Secondly mean DNA content expressed in relative units (DNA-r.u.) was computed according to an internal euploid control. Finally entropy was computed from each histogram of DNA content. Experimental design was based on a repeated measures analysis of variance with a priori orthogonal comparisons. We found that DNA content and particularly entropy are not randomly distributed. Furthermore, there was a significant difference between the two non-tumoural epithelia.