Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma. Modern Pathology (2012) 25, 1345-1353; doi: 10.1038/modpathol.2012.96; published online 8 June 2012

Studio di marcatori immunoistochimici, analisi FISH e ruolo dei microRNA nelle neoplasie gliali di basso e di alto grado

Gliomas are the most common primary brain tumours. Despite advances in surgical techniques, postoperative supportive care, radiation and adjuvant systemic therapy, the life expectancy of patients with high grade glioma has remained essentially poor. Furthermore differential diagnosis among astrocytomas, oligodendrogliomas and oligoastrocytomas is very challenging and subject to inter-observer variability. The purpose of the research was: 1) to investigate a series of high grade and low grade gliomas at gene and protein (immunohistochemistry) levels to disclose possible genetic portraits of malignancy; 2) to verify the utility of Nogo-A, Olig-2 and synaptophysin in providing a correct histological diagnosis of oligodendroglioma and to investigate a possible complementary role in selecting the best areas suitable for detecting 1p/19q codeletion using FISH analysis; 3) to study the role of microRNA in high grade gliomas. In order to obtain these goals large series of brain tumors were studied with DNA microarrays, immunohistochemistry and RT-PCR The results demonstrated that: - Overexpression of IGFBP-2 and CDC20 is highly related to glioblastomas and their immunopositivity can be useful for the identification of glioblastoma in small biopsies. - Nogo-A is the most useful and specific marker in differentiating oigodendrogliomas from other gliomas. Furthermore, using a Nogo-A driven FISH analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas. - microRNAs can be studied in paraffin embedded tissues better than in fresh tissues. A series of six microRNA, significatively deregulated in glioblastomas, may represent a genetic signature with prognostic and predictive value and could constitute candidates for novel anti-cancer therapeutics.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

A role for IGF-1R-targeted therapies in small-cell lung cancer?

Background: Small-cell lung cancer (SCLC) is an aggressive disease with a poor prognosis. The insulin-like growth factor-1 receptor (IGF-1R) is an autocrine growth factor and an attractive therapeutic target in many solid tumors, but particularly in lung cancer. Patients and Methods: This study examined tumor samples from 23 patients diagnosed with SCLC, 11 resected specimens and 12 nodal biopsies obtained by mediastinoscopy, for expression of IGF-1R using the monoclonal rabbit anti-IGF-1R (clone G11, Ventana Medical Systems, Tucson, AZ) and standard immunohistochemistry (IHC). Results: All 23 tumor samples expressed IGF-1R with a range of stain intensity from weak (1+) to strong (3+). Ten tumors had a score of 3+, 7 tumors 2+, and 6 tumors 1+. Patient survival data were available for all 23 patients. Two patients died < 30 days post biopsy, therefore, the intensity of anti-IGF-1R immunostaining for 21 patients was correlated to survival. Patients with 3+ immunostaining had a poorer prognosis (P = .003). The overall survival of patients who underwent surgical resection was significantly better (median survival not reached) than patients who were not resected (median survival, 7.4 months) (P = .006). Conclusion: IGF-1R targeted therapies may have a role in the treatment of SCLC in combination with chemotherapy or as maintenance therapy. Further studies on the clinical benefit of targeting IGF-1R in SCLC are needed.

The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids

Objective: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. Design: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. Setting: Free-living individuals associated with the University of Surrey. Subjects: Six male subjects with a mean (±sd) age of 51.2±3.6 y were recruited. Major Outcome Measures: Pre-and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. Results: Mean LPL expression values were 4.12´105 molecules of LPL mRNA per ng total RNA on the control diet and 4.60´105 molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P=0.03, R=-0.87 and R2=0.75) and with the total area under the TAG-time response curve (P=0.003, R=-0.96 and R2=0.92) in individuals. Conclusions: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Biodistribution of the radiophamarceutical sodium pertechnetate (Na99mTcO4) after massive small bowel resection in rats

To evaluate the biodistribution of sodium pertecnetate (Na99mTcO4) in organs and tissues, the morphometry of remnant intestinal mucosa and ponderal evolution in rats subjected to massive resection of the small intestine. Methods: Twenty-one Wistar rats were randomly divided into three groups of 7 animals each. The short bowel (SB) group was subjected to massive resection of the small intestine; the control group (C) rats were not operated on, and soft intestinal handling was performed in sham rats. The animals were weighed weekly. On the 30th postoperative day, 0.l mL of Na99mTcO4, with mean activity of 0.66 MBq was injected intravenously into the orbital plexus. After 30 minutes, the rats were killed with an overdose of anesthetic, and fragments of the liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, femur and brain were harvested. The biopsies were washed with 0.9% NaCl.,The radioactivity was counted using Gama Counter WizardTM 1470, PerkinElmer. The percentage of radioactivity per gram of tissue (%ATI-g) was calculated. Biopsies of the remaining jejunum were analysed by HE staining to obtain mucosal thickness. Analysis of variance (ANOVA) and the Tukey test for multiple comparisons were used, considering p<0.05 as signifi cant. Results: There were no signifi cant differences in %ATI-g of the Na99mTcO4 in the organs of the groups studied (p>0.05). An increase in the weight of the SB rats was observed after the second postoperative week. The jejunal mucosal thickness of the SB rats was signifi cantly greater than that of C and sham rats (p<0.05). Conclusion: In rats with experimentally-produced short bowel syndrome, an adaptive response by the intestinal mucosa reduced weight loss. The biodistribution of Na99mTcO4 was not affected by massive intestinal resection, suggesting that short bowel syndrome is not the cause of misleading interpretation, if an examination using this radiopharmaceutical is indicated

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Baixas doses de irradiação associadas à infusão de células da medula óssea não previnem a ocorrência da reação do enxerto versus hospedeiro após o transplante intestinal. OBJETIVO: Neste estudo foi avaliado a potencial vantagem em estender o regime imunossupressor associado a infusão de células de medula óssea do doador depletadas de células T na prevenção da reação do enxerto versus hospedeiro após o transplante intestinal. MÉTODOS: Transplante heterotópico de intestino delgado foi realizado em ratos Lewis como receptores e da como doadores, distribuídos em cinco grupos de acordo com a duração da imunossupressão, irradiação e do uso de medula óssea normal ou depletada: G1 (n=6), sem irradiação e G2 (n=9), G3 (n=4), G4 (n=5) e G5 (n=6) foram irradiados com 250 rd. Grupos1, 2, 4 e G3 e 5 foram infundidos com 100 x 10(6) células da medula normal e depletada respectivamente. Animais no G1,2,3 foram imunossuprimidos com 1mg/kg/FK506/ IM por cinco dias e G4 e cinco por 15 dias. Anticorpos monoclonais contra células CD3 e colunas magnéticas foram utilizadas para a depleção da medula óssea. Os animais foram examinados para a presença de rejeição, reação do enxerto versus hospedeiro, chimerismo e biópsias intestinais e da pele. RESULTADOS: Rejeição mínima foi observada em todos os grupos; entretanto, a reação do enxerto versus hospedeiro somente nos animais irradiados. Extensão da imunossupressão alterou a gravidade da reação nos animais dos G4 e 5. Rejeição foi a causa mortis no G1 e a reação do enxerto versus hospedeiro nos Grupos 2,3,4 e 5, não controlada com a infusão de medula óssea depletada. O chimerismo total e de células T do doador foi estatisticamente maior nos grupos irradiados em comparação ao G1. CONCLUSÃO: A extensão do regime de imunossupressão associado a baixas doses de irradiação diminui a gravidade da reação do enxerto versus hospedeiro, não abolida pelo uso de medula óssea depletada.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

Em estudo recente demonstramos que a infusão de células da medula óssea do doador após o transplante intestinal não aumentou a sobrevida do enxerto quando se utilizou series curtas de drogas imunossupressoras. OBJETIVO: Neste estudo avaliamos se a utilização de diferentes regimes de irradiação em associação com a infusão de medula óssea altera a sobrevida do enxerto e a morbidade sobre receptor. MÉTODOS: Realizou-se o transplante heterotópico de intestino delgado, utilizando-se ratos Lewis como receptores e da como doadores, imunossuprimidos com FK 506 na dose de 1mg/kg/dia por 5 dias e distribuídos em 4 grupos: G1 (n= 4), não irradiado e sem infusão de medula óssea; G2 (n= 6), G3 (n= 9) e G4 (n= 6) foram infundidos com 100 x 10(6) células de medula após o transplante. Grupos 3 e 4 foram irradiados com 250 e 400 rd respectivamente. Os animais foram examinados diariamente para a detecção de rejeição e reação do enxerto versus hospedeiro, tendo sido colhidas amostras semanais de sangue para estudos de quimerismose biopsias quinzenais da estomia. RESULTADOS: Animais nos G1 e G2 apresentaram rejeição mínima no 15º pós-operatório, enquanto a reação do enxerto versus hospedeiro foi caracterizada nos G3 e G4. Os níveis de quimerismo total e de células T foram maiores nos grupos irradiados em comparação aos não irradiados. A causa mortis nos G1 e G2 foi a rejeição enquanto que nos G3 e G4 foi a reação do enxerto versus hospedeiro. CONCLUSÃO: Concluímos que a utilização de baixas doses de irradiações retardam o aparecimento da rejeição, mas não previne a ocorrência da reação do enxerto versus hospedeiro.

Comparative Analysis of the Complications of 5347 Endomyocardial Biopsies Applied to Patients After Heart Transplantation and With Cardiomyopathies: A Single-center Study

Introduction. Endomyocardial biopsy (EMB) plays an important role in allograft surveillance to screen an acute rejection episode after heart transplantation (HT), to diagnose an unknown cause of cardiomyopathies (CMP) or to reveal a cardiac tumor. However, the procedure is not risk free. Objective. The main objective of this research was to describe our experience with EMB during the last 33 years comparing surgical risk between FIT versus no-HT patients. Method. We analyzed retrospectively the data of 5347 EMBs performed from 1978 to 2011 (33 years). For surveillance of acute rejection episodes after HT we performed 3564 (66.7%), whereas 1777 (33.2%) for CMP diagnosis, and 6 (1.0%) for cardiac tumor identification. Results. The main complications due to EMB were divided into 2 groups to facilitate analysis: major complications associated with potential death risk, and minor complications. The variables that showed a significant difference in the HT group were as follows: tricuspid Injury (.0490) and coronary fistula (.0000). Among the no-HT cohort they were insufficient fragment (.0000), major complications (.0000) and total complications (.0000). Conclusions. EMB can be accomplished with a low risk of complications and high effectiveness to diagnose CMP and rejection after HT. However, the risk is great among patients with CMP due to their anatomic characteristics. Children also constitute a risk group for EMB due to their small size in addition to the heart disease. The risk of injury to the tricuspid valve was higher among the HT group.

Quality, size, and composition of pediatric endobronchial biopsies in cystic fibrosis

BACKGROUND: Studies on airway remodeling in children with cystic fibrosis (CF) may be hampered by difficulty in obtaining evaluable endobronchial biopsy specimens because of large amounts of mucus and inflammation in the CF airway. We prospectively assessed how the quality of biopsy specimens obtained from children with CF compare with those from children with other airway diseases. METHODS: Fiberoptic bronchoscopy with endobronchial biopsy was performed in 67 CF children (age range, 0.2 to 16.8 years), 34 children with wheeze/asthma (W/A), and 64 control children with chronic respiratory symptoms. Up to three biopsy specimens were taken and stained with hematoxylin and eosin. Biopsy specimen size and structural composition were quantified using stereology. RESULTS: At least one evaluable biopsy specimen was obtained in 72% of CF children, in 79% of children with W/A, and in 72% of control subjects (difference was not significant). The use of large biopsy forceps (2.0 mm) rather than small biopsy forceps (1.0 mm) [odds ratio (OR), 5.8; 95% confidence interval (CI), 1.1 to 29.8; p = 0.037] and the number of biopsy specimens taken (odds ratio, 2.6; 95% confidence interval, 1.3 to 5.2; p = 0.006) significantly contributed to the success rate. Biopsy size and composition were similar between groups, except that CF children and those patients with W/A had a higher percentage of the biopsy specimen composed of muscle than did control subjects (median 6.2% and 9.7% vs 0.9%, respectively; p = 0.002). CONCLUSIONS: Biopsy size and quality are adequate for the study of airway remodeling in CF children as young as 2 months of age. Researchers should use large forceps when possible and take at least two biopsy specimens per patient. An increased airway smooth muscle content of the airway mucosa may contribute to the pathophysiology of CF lung disease.