Pre-embedding of small biopsies using a Paraform gel system in a high throughput pathology laboratory.

In histology, the correct handling and orientation of small/thin biopsies is often crucial for diagnosis. Automation is progressively growing and modifying the routine work in the histopathology laboratories, providing new chances for quality improvement and workload optimization. We have tested the use of Paraform orientation gels together with an automated embedding system for processing small/thin biopsies, first skin, but also other tissue/organ biopsies. The study aimed to assess the benefits and challenges of routinely using orientation gels in a high throughput pathology laboratory. Gel introduction required a short training of the pathologists, including trainees, at grossing; it did not cause significant delay at grossing, interference with embedding, or microtome steps, whereas re-do inclusions and re-cut slides were significantly reduced. In conclusion, orientation gel and automatic embedding constituted an efficient system for small/thin biopsies that had to be correctly placed and orientated, allowing the re-modeling of technicians' workflow and very safe handling of small/thin biopsies that were not manipulated further after grossing.

Epidermal growth factor receptor and K-RAS mutations in 411 lung adenocarcinoma: a population-based prospective study.

Targeting the epidermal growth factor receptor has played a central role in advanced non-small cell lung cancer research, treatment, and patient outcomes over the last several years; however, a number of questions about this approach remain to be addressed. Through the Istituto Toscano Tumori and the Italian Association of Women Against Lung Cancer Project, we collected 411 lung adenocarcinomas from several clinical centers in Tuscany. Mutations were assessed by sequencing exons 18-21 of the epidermal growth factor receptor gene, and by restriction fragment length polymorphism analysis of codons 12 and 13 of the K-RAS gene. Epidermal growth factor receptor mutations (12.6%) were more frequently observed in females (p<0.0001), in non-smokers (p=0.005), and in the presence of bronchioloalveolar features (p=0.0004). K-RAS mutations (17.9%) were more frequent in males (p=0.0007) and were associated with smoking habits (p=0.005). Epidermal growth factor receptor and K-RAS mutations were mutually exclusive (p=0.001). We focused on 21 female patients with advanced/metastatic lung adenocarcinoma who received gefitinib 250 mg/day (expanded access) or erlotinib 150 mg/die as second/third-line therapy; partial response was associated with classic epidermal growth factor receptor mutations (p=0.006) and with a non-smoking history (p=0.02). None of the female patients with partial response and/or stable disease showed K-RAS alterations. Although the data obtained in our study have yet to be analyzed and confirmed with a larger number of patients treated with tyrosine kinase inhibitors, they should provide useful information for targeted therapy, in particular for non-smoking female lung cancer patients.

Different estrogen receptor beta expression in distinct histologic subtypes of lung adenocarcinoma.

Adenocarcinoma is becoming the most common histologic type of lung cancer in both sex. Although most cases are seen in smokers, it develops more frequently than other histologic types in individuals who have never smoked. This evidence suggests that other putative etiologic factors, such as sex hormones, need to be investigated. Several subtypes of lung adenocarcinoma have been recently described with distinct clinicopathologic features and prognostic implications. The purpose of this study is to investigate the role of estrogen receptor beta in lung adenocarcinoma, with particular attention paid to its different histologic subtypes. Nuclear estrogen receptor beta expression was evaluated by immunohistochemistry in 112 lung adenocarcinomas, including both "single subtype" and "mixed subtype" samples. Using a 2-level (high/low) score system, estrogen receptor beta expression was high in most (75%) adenocarcinomas and turned out to be strongly related to the histologic subtypes. In fact, estrogen receptor beta expression was low or negative in 68.2% of solid subtypes, whereas it was high in 76.5% of nonmucinous bronchioloalveolar, in 69.4% of acinar, and in 61.2% of papillary patterns (P = .00004). Furthermore, a strong association between estrogen receptor beta expression and tumor histologic grade was observed: estrogen receptor beta was highly expressed predominantly in well- and moderately differentiated tumors (P = .0014). In conclusion, estrogen receptor beta expression has distinct patterns in lung adenocarcinoma, suggesting a specific role for estrogen receptor beta in the pathogenesis of different histologic subtypes of this type of cancer. Moreover, loss of estrogen receptor beta expression in poorly differentiated (G3) tumors could represent a crucial step in the dedifferentiation process of lung adenocarcinoma.

Mutational analysis in cytological specimens of advanced lung adenocarcinoma: a sensitive method for molecular diagnosis.

INTRODUCTION: The discovery that somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with sensitivity to the EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinomas, whereas Kras mutations are associated with resistance, has generated excitement among both clinicians and researchers studying non-small cell lung cancer (NSCLC). Mutational analysis may soon be very useful in choosing among a wide range of targeted therapies to individualize treatment to tumor characteristics. This analysis would be even more useful in patients with advanced NSCLC, in whom cytological specimens are often the only material available. METHODS: We analyzed 23 archived cytologic specimens of advanced/metastatic lung adenocarcinomas for mutations in EGFR exons 18 to 21, and Kras exon 2. RESULTS: Our data show that our cytological specimens were perfectly adequate for the molecular analysis of EGFR and Kras mutations. EGFR TK domain mutations were found in three cases (13.04%) and were associated with both female gender (p = 0.02) and a nonsmoking history (p = 0.008). Moreover, we explored the relationship between EGFR mutation status and the presence of Kras mutations. Kras mutations involving codon 12 in exon 2 were found in 5 (21.73%) of the 23 adenocarcinomas and were associated, where known, with smoking habits. We never found EGFR alterations in tumors with Kras mutations. CONCLUSIONS: Our results provide oncologists with a highly accurate laboratory method to identify biological predictors of the efficacy of different therapies, and they may have an important impact on clinical practice. This method may be particularly useful in patients with advanced/metastatic NSCLC.

Galectin-3 is highly expressed in nonencapsulated papillary thyroid carcinoma but weakly expressed in encapsulated type; comparison with Hector Battifora mesothelial cell 1 immunoreactivity.

The histologic diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) may be troublesome, especially in its encapsulated form. We evaluated the expression of galectin-3 (gal-3) and Hector Battifora mesothelial cell (HBME-1) in 200 formalin-fixed thyroid tissues with diagnosis of classical variant of papillary thyroid carcinoma or FVPTC, encapsulated or with infiltrative growth, with or without lymph node metastasis. All cases of classical variant of papillary thyroid carcinoma were consistently positive for gal-3; similar results have been obtained by using HBME-1. Interestingly, the invasive type of FVPTC, with or without metastasis, was strongly positive for gal-3 (78.2% and 96%, respectively), whereas only 46.8% of encapsulated FVPTCs without metastasis showed immunostaining for this marker. In the latter group, the HBME-1 expression achieved a significantly higher percentage of positivity (87%). Surprisingly, gal-3 immunodetection showed negative results in 4 encapsulated FVPTCs, despite the strong immunoreactivity in corresponding metastasis. Our data suggest that gal-3 immunodetection alone is not able to support the diagnosis of encapsulated FVPTCs.

Osteopontin expression and prognostic significance in non-small cell lung cancer.

PURPOSE: The survival rate of non-small cell lung cancer patients is very low, and knowledge of predictors of outcome is inadequate. To improve the curability of lung cancer, we need to identify new specific molecules involved in tumorigenesis and progression. The purpose of this study was to better define the role of osteopontin in non-small cell lung cancer biology by determining its prognostic significance. EXPERIMENTAL DESIGN: Osteopontin expression was evaluated by immunohistochemistry, as percentage of neoplastic cells with cytoplasmic immunoreactivity, in a wide series of patients with stage I-IIIA non-small cell lung cancer (207 cases). The median value of this series (20% of positive cells) was used as the cutoff value to distinguish tumors with low (<20%) from tumors with high (> or =20%) osteopontin expression. RESULTS: Taking the series of patients as a whole (207 cases), osteopontin expression was associated with neither overall survival (P = 0.14) nor disease-free survival (P = 0.074). However, among patients with at least 6 years of follow-up (163 cases), 6-year overall survival and disease-free survival were significantly reduced if osteopontin expression was high (P = 0.0085 for overall survival, P = 0.0023 for disease-free survival). Moreover, a statistically significant correlation between high levels of osteopontin and shorter overall survival (P = 0.034) and disease-free survival (P = 0.011) in patients with stage I tumors (136 cases) was shown. CONCLUSIONS: Our results support the hypothesis of an association between high osteopontin expression and poor survival of patients with stage I non-small cell lung cancer, suggesting that osteopontin could be a candidate target for cancer therapy.

Distal embolization during primary angioplasty: histopathologic features and predictability.

BACKGROUND: Distal embolization during primary percutaneous coronary interventions (PCIs) may affect myocardial reperfusion. We evaluated the prevalence and features of embolization during primary PCI and its relationship with clinical and angiographic variables. METHODS: Forty-six consecutive patients with acute myocardial infarction underwent primary PCI with a filter-based distal protection device. Histopathologic analysis was performed on retrieved embolic fragments, assessing the presence and relative amount of fibrin, necrosis, lipid droplets, collagen, mucopolysaccharides, and leukocytes, as well as the total debris volume. Such variables were related to baseline clinical and angiographic variables. RESULTS: Embolic material was recovered in 41 (89%) of 46 cases, with a mean total debris volume of 1.2 +/- 2.2 mm3. Prevalent histopathologic patterns were organized thrombus (47%), fresh thrombus (29%), and plaque fragments (24%). At multivariate analysis, none of the baseline clinical variables considered significantly predicted the total debris volume. Among angiographic variables, angiographic signs of high thrombus burden (cut-off coronary occlusion pattern or large intracoronary minus image) independently predicted the total debris volume at multivariate analysis (odds ratio 15.8, P < .005). Compared with its nonuse, abciximab did not affect the total number and the mean total volume of embolized material (15 +/- 16 vs 10 +/- 8 fragments, 1.5 +/- 2.5 vs 1.0 +/- 1.9 mm3, respectively, for both P > .20), or its qualitative composition. CONCLUSIONS: Distal embolization occurs in most patients during primary PCI and mainly consists of plaque fragments and partially organized thrombi, which are likely to be scarcely responsive to antiplatelet drugs. Baseline angiographic signs of a high thrombus burden are the only significant predictors of the extent of distal embolization.

Immunohistochemical and molecular study of radiation-induced multiple meningiomas with pleural and pulmonary metastasis.

In the present study, the telomerase activity and the putative alterations of genes involved in cell-cycle control (p53, Fas and pRb) were investigated in a radiation-induced meningioma with multiple recurrences and pleural-pulmonary metastases (the patient, a 34-year-old male, had a history of carcinoma of the tongue of testicular lymphocytic lymphoma). Expression of VEGF and vasculature pattern were also studied. Expression of VEGF, pRb and p53 were evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded samples of the tumor. VEGFmRNA was determined by competitive PCR. Fas, FasL and hTERT were evaluated by RT-PCR. Telomerase activity was examined by the TRAP assay. An intense vascularization was observed, supported by high expression of VEGFmRNA (isoforms 121 and 165). pRb and p53 were overexpressed. Fas was undetectable with PCR, whereas FasL was positive. Furthermore, the lesion showed an elevated telomerase activity (TPG, 22), according to the high expression of hTERT. These findings emphasized that even among generally benign neoplasms, such as meningiomas, some highly malignant tumors may develop, as in our case, in which several mechanisms were activated in the cancer progression to guarantee the immortalization of cellular clones (angiogenic phenomenon, activation of telomerase and of anti-apoptotic mechanisms) and the blood spread. Thus, the data illustrate the importance of searching for genetic aberrations (which are a hallmark of malignancy) in meningiomas, as predictive and reliable factors of the possibility to recur and to metastasize.

Expression and mutational status of c-kit in small-cell lung cancer: prognostic relevance.

PURPOSE: The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family. Kinase activity has been implicated in the pathophysiology of many tumors, including small-cell lung carcinoma (SCLC). Autocrine or paracrine activation of c-kit by its ligand has been postulated for lung cancer, but this receptor can also be activated by mutations of the c-kit gene. We examined c-kit expression and mutational status in SCLC to verify its putative expression and genetic alterations, as well as its eventual prognostic impact. EXPERIMENTAL DESIGN: We studied 60 SCLC samples to determine the mutations of the coding region of the gene; the exons 9 and 11 were analyzed by PCR-single-strand conformational polymorphism and automated sequencing. Moreover, c-kit expression was evaluated in 55 samples by immunohistochemical method. RESULTS: Expression of c-kit was demonstrated in about 40% of SCLC samples. Two mutations in exon 9 and three mutations in exon 11 were found. Kaplan-Meier analysis revealed no prognostic significance of c-kit expression for survival. CONCLUSIONS: In our series, the expression of c-kit and its mutational status failed to appear relevant or to have a significant impact on survival; this makes the therapeutic approach with an inhibitor of tyrosine kinase more difficult in SCLC until a sure demonstration of c-kit implication is obtained for this tumor.

Applications of tissue microarray technology in immunohistochemistry: a study on c-kit expression in small cell lung cancer.

Tissue microarray technology allows the immediate evaluation of molecular profiles of numerous different tissues, with savings of money and time. It was created for rapid, large-scale molecular studies, and the main concern regarding its possible broad acceptance is that the analysis of tissue microarrays instead of whole tissue sections may lead to false negative or positive results because of tissue heterogeneity. In the present study, we analyzed in 54 small cell lung cancers, by immunohistochemistry, the expression of the antigen c-kit, which seems to be important in these neoplasms' tumorigenesis, and compared the staining obtained on whole sections with that of the corresponding tissue microarrays. Although c-kit expression of the whole sections agreed with that of the corresponding biopsies in many cases, the correlation between whole sections and all the companion nonlost single cores or their mean value turned out to be highly significant only if the 36 double negatives (ie, both whole sections and companion tissue microarrays negative) were included (P <0.0001). In fact, if only cases positive to at least 1 of the tests (i.e. whole sections or corresponding tissue microarrays positive) were considered, the correlation was not significant (P=0.055). Tissue microarrays showed a good specificity (94.2% for all single cores and 92.3% for their mean value) but a rather poor sensitivity (respectively, 69.4% and 71.4%). Moreover, a high percentage (13.4%) of cores was lost, and this loss was not random. To sum up, in our experience, tissue microarray technology cannot be a substitute for whole sections in clinical diagnosis of individual cases.

Galectin-3 and oncofetal-fibronectin expression in thyroid neoplasia as assessed by reverse transcription-polymerase chain reaction and immunochemistry in cytologic and pathologic specimens.

Oncofetal fibronectin (onfFN) and galectin-3 (Gale-3) have been proposed as possible tools for the preoperative diagnosis of thyroid carcinomas, based on the finding that the expression of both onfFN and Gale-3 are significantly increased in papillary and anaplastic carcinomas, compared to normal thyroid tissues and follicular adenomas. In this study we analyzed the expression of these markers by immunochemical and molecular analysis of benign and malignant thyroid tumors. Sixty-five thyroid nodules, consisting of 20 follicular adenomas (FAs) and 45 papillary thyroid carcinomas (PTCs) at final histology were examined. At the molecular level, among the 45 PTCs, 44 (97.8%) showed a variable level of onfFN mRNA, while 8 of the 20 (40%) adenomas expressed the same marker. Similar results have been found analyzing Gale-3 expression: 97.8% of PTC and 55% of FAs were positive for this marker. Immunohistochemistry (IHC) for Gale-3 was positive in 42 of 45 (93.3%) PTC tissues. Staining was invariably confined to the cytoplasm, with a homogeneous distribution in the large majority of the neoplastic cells. The 3 negative cases (6.7%) were represented by 2 classic variants of PTC and 1 follicular variant of PTC. Eighteen of the 20 (90.0%) adenomas stained negative for Gale-3. A significant association was found between positive staining and malignant phenotype (p < 0.0001). Gale-3 protein expression was also performed on samples obtained by ex vivo fine-needle aspiration (FNA) in 35 PTCs by immunocytochemistry (ICC). Immunoreactivity was present in 32 (91.0%) and negative in 3 (8.8%) cases. With the exception of 1 case (negative by ICC and positive by IHC), ICC and IHC were fully concordant. In conclusion, our results indicate that a search for Gale-3 protein overexpression by IIC or ICC, but not by reverse transcription-polymerase chain reaction (RT-PCR), may yield an additional marker of malignant potential of thyroid nodular lesions, and may be a useful adjunct to the currently available diagnostic tools for the preoperative diagnosis of malignant thyroid tumors.

The role of somatostatin in vasogenic meningioma associated brain edema.

Many tumors, including meningiomas, express somatostatin receptors, suggesting the application of somatostatin analogues for therapy and diagnosis. Sixty percent of meningiomas are associated with perilesional edema, whose development seems to be related to the vascular endothelial growth factor, although it requires an efficient pial blood supply. However, in several neoplastic models, other mediators seem to cooperate with vascular endothelial growth factor in regulating angiogenesis. We evaluated somatostatin receptors (sst2) in relation to the possibility that somatostatin analogues may influence vascular endothelial growth factor production with reduction of edema. Of 35 studied meningiomas, 21 presented peritumoural edema. Vascular endothelial growth factor, microvascular density and pial blood supply were significantly related to the edema (P = 0.0001, P = 0.0001, P = 0.0005). Similarly, a relation was found between sst2 and microvascular density (r = 0.58, P < 0.001) and between sst2 and vascular endothelial growth factor expression (P = 0.03). This suggests that somatostatin analogues may be relevant for the treatment of meningiomas.

CD34 microvessel density and VEGF expression in basal and squamous cell carcinoma.

Angiogenesis is a central process in the growth of solid tumors. The purpose of our study was to analyze the angiogenic pattern in squamous and basal cell carcinomas and to point out differences in microvessel density that could explain their different biological behaviour. Thirty-nine skin tumors (26 basal and 13 squamous cell carcinomas) were analyzed. In all samples, the microvessels density (MVD) and the levels of vascular endothelial growth factor mRNA (VEGFmRNA) were analyzed, together with the inter-relationship between these two variables. Using the median value of the entire series (33 vessels per 2.22 mm2), tumors with low and high MVD were identified. The majority of cancers with high vascularization belonged to the squamous histotype (12 of 39), while 19 of the 26 basal cell carcinomas showed a lower number of microvessels than the median value (p = 0.0001). The median value of VEGFcDNA quantitation allowed us to distinguish tumors with high VEGF expression (> 470 molecules cDNA) from those with low (< or = 470 molecules) VEGF expression: 20 of the 26 basal cell carcinomas showed low VEGF expression, while 11 of the 13 squamous cell carcinomas showed high VEGFcDNA levels (p = 0.0003). Moreover, a significant association between a high microvessel density and high VEGFmRNA levels (p = 0.006) was found. Furthermore, when studying VEGF expression by immunohistochemistry, we obtained similar results and noted a correlation with VEGFmRNA expression (p < 0.0001). The association between high vascularization, high VEGF levels, and squamous cell histotype suggests the possible role of neoangiogenesis in determining the more aggressive biological behaviour of this type of cancer.