Interobserver variability for the WHO classification of pulmonary carcinoids.

Pulmonary carcinoids are neuroendocrine tumors histopathologically subclassified into typical (TC; no necrosis, <2 mitoses per 2 mm) and atypical (AC; necrosis or 2 to 10 mitoses per 2 mm). The reproducibility of lung carcinoid classification, however, has not been extensively studied and may be hampered by the presence of pyknotic apoptosis mimicking mitotic figures. Furthermore, prediction of prognosis based on histopathology varies, especially for ACs. We examined the presence of interobserver variation between 5 experienced pulmonary pathologists who reviewed 123 originally diagnosed pulmonary carcinoid cases. The tumors were subsequently redistributed over 3 groups: unanimously classified cases, consensus cases (4/5 pathologists rendered identical diagnosis), and disagreement cases (divergent diagnosis by ≥2 assessors). κ-values were calculated, and results were correlated with clinical follow-up and molecular data. When focusing on the 114/123 cases unanimously classified as pulmonary carcinoids, the interobserver agreement was only fair (κ=0.32). Of these 114 cases, 55% were unanimously classified, 25% reached consensus classification, and for 19% there was no consensus. ACs were significantly more often in the latter category (P=0.00038). The designation of TCs and ACs by ≥3 assessors was not associated with prognosis (P=0.11). However, when disagreement cases were allocated on the basis of Ki-67 proliferative index (<5%; ≥5%) or nuclear orthopedia homeobox immunostaining (+; -), correlation with prognosis improved significantly (P=0.00040 and 0.0024, respectively). In conclusion, there is a considerable interobserver variation in the histopathologic classification of lung carcinoids, in particular concerning ACs. Additional immunomarkers such as Ki-67 or orthopedia homeobox may improve classification and prediction of prognosis.

Deletions of 11q22.3-q25 are associated with atypical lung carcinoids and poor clinical outcome.

Carcinoids are slow-growing neuroendocrine tumors that, in the lung, can be subclassified as typical (TC) or atypical (AC). To identify genetic alterations that improve the prediction of prognosis, we investigated 34 carcinoid tumors of the lung (18 TCs, 15 ACs, and 1 unclassified) by using array comparative genomic hybridization (array CGH) on 3700 genomic bacterial artificial chromosome arrays (resolution ≤1 Mb). When comparing ACs with TCs, the data revealed: i) a significant difference in the average number of chromosome arms altered (9.6 versus 4.2, respectively; P = 0.036), with one subgroup of five ACs having more than 15 chromosome arms altered; ii) chromosomal changes in 30% of ACs or more with additions at 9q (≥1 Mb) and losses at 1p, 2q, 10q, and 11q; and iii) 11q deletions in 8 of 15 ACs versus 1 of 18 TCs (P = 0.004), which was confirmed via fluorescence in situ hybridization. The four critical regions of interest in 45% ACs or more comprised 11q14.1, 11q22.1-q22.3, 11q22.3-q23.2, and 11q24.2-q25, all telomeric of MEN1 at 11q13. Results were correlated with patient clinical data and long-term follow-up. Thus, there is a strong association of 11q22.3-q25 loss with poorer prognosis, alone or in combination with absence of 9q34.11 alterations (P = 0.0022 and P = 0.00026, respectively).

The clinical value of lymphatic micrometastases in patients with non-small cell lung cancer.

INTRODUCTION: In early stage non-small cell lung cancer (NSCLC), presence of lymphatic micrometastases and isolated tumor cells, primarily detected by immunohistochemistry, is suggested to be a prognostic factor. However, there is no consensus whether immunohistochemistry should be used routinely in lymph node assessment.The goal of our study was to determine whether recurrent disease is associated with the presence of lymphatic micrometastases and/or isolated tumor cells, at the time of the lung resection. METHODS: We retrospectively analyzed the prevalence of lymphatic micrometastases and/or isolated tumor cells in two groups of patients, who underwent a curative resection for early stage NSCLC. Group I had a follow-up of 5 years without recurrent disease. Group II consisted of a matched group of patients with recurrent disease. Patients were originally classified as having negative mediastinal lymph nodes.All lymph nodes obtained by mediastinoscopy and thoracotomy were re-examined by serial sectioning and immunohistochemistry. RESULTS: Micrometastases and/or isolated tumor cells were found in one of 16 patients in group I, which was significantly different from six of 16 patients in group II. (Fisher exact test, 4.6; p, 0.04; risk ratio, 2.4).Serial sectioning and immunohistochemistry did not change N-stage for the single patient in group I, in contrast to all six patients in group II. CONCLUSION: Presence of lymphatic micrometastases and/or isolated tumor cells is associated with distant recurrence in patients with early stage NSCLC. We recommend the routine use of serial sectioning and immunohistochemistry in lymph node assessment to improve the accuracy of staging.

Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.

AIMS: To test the hypothesis that the published morphological criteria permit reliable segregation of small cell carcinoma of the lung (SCLC) and large cell neuroendocrine carcinoma (LCNEC) cases by determining the interobserver variation. METHODS AND RESULTS: One hundred and seventy cases of SCLC, LCNEC and cases diagnosed as neuroendocrine lung carcinoma before LCNEC had been established as a diagnostic category were retrieved from the archives of the assessor's institutes. A representative haematoxylin and eosin section from each case was selected for review. Batches of cases were circulated among nine pathologists with a special interest in pulmonary pathology. Participants were asked to classify the cases histologically according to the 2004 World Health Organization (WHO) criteria. The diagnoses were collected and kappa values calculated. Unanimity of diagnosis was achieved for only 20 cases; a majority diagnosis was reached for 115 cases. In 35 cases no consensus diagnosis could be reached. There was striking variability amongst assessors in diagnosing SCLC and LCNEC. The overall level of agreement for all cases included in this study was fair (kappa=0.40). CONCLUSIONS: Using non-preselected cases, the morphological WHO criteria for diagnosing SCLC and LCNEC leave room for subjective pathological interpretation, which results in imprecise categorization of SCLC and LCNEC cases.

Circulating DNA is a non-invasive prognostic factor for survival in non-small cell lung cancer.

INTRODUCTION: Circulating plasma DNA is present in a considerably higher concentration in lung cancer patients than in controls. Conflicting data are reported about circulating DNA as a prognostic factor. The aim of this study was to prospectively analyse the relationship of circulating plasma DNA with overall survival (OS) of previously untreated non-small cell lung cancer (NSCLC) patients. METHODS: 46 untreated NSCLC patients and 21 controls with a follow-up time of 6.5 years were analyzed. Quantification of baseline circulating plasma DNA was performed by a real-time quantitative polymerase chain reaction (qPCR) targeting the human beta-globin gene. Survival analysis was performed using the Kaplan-Meier method and compared with a Cox-regression analysis. RESULTS: The median DNA concentration of the patients who died (87%) was significantly higher compared to the patients that survived at the end of follow-up (55ng/ml versus 23ng/ml, p=0.02). In patients with higher DNA concentration overall survival was significantly worse. In this study no relation of DNA concentration with tumour characteristics, age, gender or pulmonary inflammatory conditions was found. CONCLUSION: In this study a high circulating plasma DNA concentration at time of diagnosis in NSCLC patients was a prognostic factor for poorer survival. Circulating DNA may be used as a non-invasive biomarker to refine the prognostic profile in NSCLC patients.

Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma.

BACKGROUND: To the authors' knowledge, there are no reliable markers able to identify patients with nonsmall cell lung cancer (NSCLC) that will develop metastases to the brain. The authors investigated associations between immunohistochemical markers and the development of brain metastases in patients with NSCLC. METHODS: This was a hospital-based, case-control study of patients who were newly diagnosed with NSCLC between 1989 and 2003, developed brain metastases, and had pathology material available from both the primary NSCLC and the brain metastases. These patients were compared with a control group of patients who had NSCLC and no evidence of brain metastases. NSCLC was examined for expression levels of Ki-67, caspase-3, vascular endothelial growth factor A (VEGF-A), VEGF-C, E-cadherin, and epidermal growth factor receptor (EGFR) in 54 surgical pathology specimens using immunohistochemistry, and associations were evaluated between those markers and the development of brain metastases. RESULTS: Brain metastases developed after a median of 12.5 months (range, 1.7-89.4 months) after the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients with NSCLC who had primary tumors with high Ki-67 levels (adjusted odds ratio [OR] of 12.2; 95% confidence interval [95% CI], 2.4-70.4 [P < .001]), low caspase-3 expression (adjusted OR of 43; 95% CI, 5.3 to >100 [P < .001]), high VEGF-C expression (adjusted OR of 14.6; 95% CI, 2.0 to >100 [P < .001]), and low E-cadherin (adjusted OR of 3.6; 95% CI, 0.9-16.4 [P = .05]). No significant risk was associated with VEGF-A or EGFR expression. High Ki-67 expression also was associated with a shorter overall survival (P = .04). CONCLUSIONS: The results of the current study indicated that patients with NSCLC who had high Ki-67 expression, low caspase-3 expression, high VEGF-C expression, and low E-cadherin expression in their tumors may benefit from close surveillance because they may have an increased risk of developing brain metastases.

Risk factors for non-sentinel lymph node metastases in patients with breast cancer. The outcome of a multi-institutional study.

BACKGROUND: In this multi-institutional prospective study, we evaluated whether we could identify risk factors predictive for non-sentinel lymph node (non-SN) metastases in breast cancer patients with a positive sentinel lymph node (SN). METHODS: In this multi-institutional study, 541 eligible breast cancer patients were included prospectively. RESULTS: The occurrence of non-SN metastases was related to the size of the SN metastasis (P = .02), primary tumor size (P = .001), and lymphovascular invasion (P = .07). The adjusted odds ratio was 3.1 for SN micro-metastasis compared with SN isolated tumor cells, 4.0 for SN macro-metastasis versus SN isolated tumor cells, 3.1 for tumor size (>3.0 cm compared with 3.0 cm, and with vessel invasion. CONCLUSION: We identified three predictive factors for non-SN metastases in breast cancer patients with a positive SN: size of the SN metastasis; primary tumor size; and vessel invasion. We were not able to identify a specific group of patients with a positive SN in whom the risk for non-SN metastases was less than 5%.

A prospective study of the timing and cost-effectiveness of bronchial washing during bronchoscopy for pulmonary malignant tumors.

STUDY OBJECTIVES: The value of obtaining washings during fiberoptic bronchoscopy in the workup of lung cancer is controversial. Moreover, the optimal timing of washing relative to biopsy and brushing is not known. In this study, the diagnostic yields of washings before and after biopsy and brushings were compared. The different diagnostic strategies were assessed in terms of yield and costs. DESIGN: A prospective study performed from 2001 to 2003 in a secondary care medical center. MEASUREMENTS AND RESULTS: Two hundred twenty-one patients underwent flexible bronchoscopy, and the diagnostic yield of washings before biopsy and brushing (strategy I) and after biopsy and brushing (strategy II) specimens were assessed. Using the known probabilities and costs for various bronchoscopic procedures, the expected utility of a number of diagnostic strategies was estimated. Patients (147 men and 74 women) were included in the study in whom a definite cytologic or histologic diagnosis of pulmonary malignancy had been made. The diagnostic yield of strategy I was 72% for visible tumors and 36% for nonvisible tumors. For strategy II, the diagnostic yield was 74% for visible tumors and in 42% for nonvisible tumors. The comparison of strategies I and II for both visible and nonvisible tumors revealed that 176 cases were concordant (80%); in 19 cases (9%) the cytologic analysis of washings in strategy I was positive for malignancy and negative in strategy II. In 26 cases (12%) washings in strategy II were positive and negative in strategy I (p = 0.37). An analysis of the diagnostic yield of both washings in visible tumors and nonvisible tumors showed no significant difference. In 13 patients, a diagnosis of malignancy was established only by washings (6%). Confining the laboratory investigations of washings or brush samples to those cases in which the initial findings of the biopsies are negative (the two-stage procedure) is more cost-effective than examining all biopsy, brushing, and washing specimens. In patients with visible tumors, brushing or washing in addition to biopsy is equally cost-effective; in patients with nonvisible tumors, biopsy combined with washing is the preferred option. CONCLUSIONS: No difference in the diagnostic yield could be demonstrated for washings before or after biopsies and brushings. Although the additional diagnostic yield of washing and brushing during bronchoscopy is relatively low, it is cost-effective to use these procedures in the diagnostic workup of patients who are clinically suspected of having a pulmonary malignancy.

Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity.

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.

Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.

Mitogen-activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients.

PURPOSE: An increase in the activity of the mitogen-activated protein kinases (MAPKs) has been correlated with a more malignant phenotype in several tumor models in vitro and in vivo. A key regulatory mechanism of the MAPKs [extracellular signal-regulated kinase (ERK); c-jun NH(2)-terminal kinase (JNK); and p38] is the dual specificity phosphatase CL100, also called MAPK phosphatase-1 (MKP-1). This study was designed to examine the involvement of CL100/MKP-1 and stress-related MAPKs in lung cancer. EXPERIMENTAL DESIGN: We assessed the expression of CL100/MKP-1 and the activation of the MAPKs in a panel of 18 human cell lines [1 primary normal bronchial epithelium, 8 non-small cell lung cancer (NSCLC), 7 small cell lung cancer (SCLC), and 2 carcinoids] and in 108 NSCLC surgical specimens. RESULTS: In the cell lines, CL100/MKP-1 expression was substantially higher in NSCLC than in SCLC. P-ERK, P-JNK, and P-p38 were activated in SCLC and NSCLC, but the degree of their activation was variable. Immunohistochemistry in NSCLC resection specimens showed high levels of CL100/MKP-1 and activation of the three MAPK compared with normal lung. In univariate analysis, no relationship was found among CL100/MKP-1 expression and P-ERK, P-JNK, or P-p38. Interestingly, high CL100/MKP-1 expression levels independently predicted improved survival in multivariate analysis. JNK activation associated with T(1-2) and early stage, whereas ERK activation correlated with late stages and higher T and N. Neither JNK nor ERK activation were independent prognostic factors when studied for patient survival. CONCLUSIONS: Our data indicate the relevance of MAPKs and CL100/MKP-1 in lung cancer and point at CL100/MKP-1 as a potential positive prognostic factor in NSCLC. Finally, our study supports the search of new molecular targets for lung cancer therapy within the MAPK signaling pathway.

DNA microarray format for detection and subtyping of human papillomavirus.

A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3' end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities.

Microarray as a model for quantitative visualization chemistry.

For visualization of proteins or nucleic acids, direct and indirect in situ fluorescence and absorption methods (immunohistochemistry and cytochemistry) have existed for many years. The authors describe a new experimental approach using microarray as a model to quantitatively compare both visualization methods. The spots obtained with the microarray robot had a progressive twofold decrease in concentrations and are used as objects with known amounts of DNA. Subsequent hybridization resulted in a direct fluorescence (DF) label or in hapten for indirect fluorescence (IF) and absorption. The results show that the image of the object in the IF method is larger than that in the DF method because of an edge effect, with stronger staining at the circumference. This leads to a higher plateau level and an 8- to 10-fold reduction in the detection threshold for IF compared with DF. These features are especially useful for one-color DNA-related microarray analysis, such as single nucleotide polymorphism, loss of heterozygosity, and mutation analysis, provided that the spots are not designed directly adjacent to each other, so that the edge effect is taken into account. The slope of the linear range for the IF method is much steeper than for the DF method, pointing to a narrow dynamic range in immunohistochemistry. It is noteworthy that the detection limit for absorption images after indirect immunoenzyme visualization is lower than for the DF images. The indirect immunohistochemistry semiquantitative absorption signal was at least similar compared with the DF fluorescence. In conclusion, an explanation for the difficulties experienced in quantitative immunohistochemistry is provided, and the data emphasize that in general, for daily pathology, semiquantitative patterns should suffice. Indirect labeling of DNA has useful characteristics for application in microarray analyses because of the large signal enhancement.

Diagnostic yield of transbronchial histology needle aspiration in patients with mediastinal lymph node enlargement.

BACKGROUND: Transbronchial needle aspiration (TBNA) is a safe, minimally invasive technique to assess the mediastinal spread of lung cancer. Excellent results have been published by experts. However, little information is available about the diagnostic yield of TBNA with the histology needle in a non-expert center. OBJECTIVES: The aim of this study is to assess the diagnostic yield of histology TBNA in the workup of suspected lung cancer. METHODS: In a non-university teaching hospital, TBNA data from patients diagnosed with lung cancer between June 1998 and July 2000 were analyzed retrospectively. TBNA had been performed by six different bronchoscopists in patients eligible for surgery with accessible N2 and N3 lymph nodes on computed tomography of the chest during the workup of an undefined mass. Cytology and histology specimens were obtained with the same 19-gauge needle. TBNA results were considered to be diagnostic if cytologic or histologic examination revealed a malignant lesion or non-malignant lymphoid cells. However, TBNA outcome was called non-diagnostic if no representative cells were obtained. RESULTS: From a group of 264 consecutive lung cancer patients, 106 (40%) patients were eligible for TBNA. In 79%, TBNA was diagnostic in cytology and/or histology specimens. Malignancy was demonstrated in 59% (63/106). In only 32/106 patients (30%), a histologic core of tissue could be sampled. In 87.5% of these patients (28/32), TBNA was diagnostic. For cytology only, this number was slightly lower (75%, 56/74). In 12 cases, diagnostic TBNA was verified by mediastinoscopy: these diagnoses were concordant. The sensitivity is 65% if all non-confirmed cases are considered false negative. Ten mediastinoscopies were avoided because TBNA demonstrated contralateral N2 (= N3) disease. The routine use of TBNA during bronchoscopy in suspected N2 disease is a cost-effective procedure, as the total additional costs of TBNA (9,540 EUR) were lower than the costs of 10 avoided mediastinoscopies (15,500 EUR). No complications were observed. CONCLUSION: The diagnostic yield of TBNA relied mainly on cytology specimens, despite the use of a histology needle. Representative histology specimens could only be obtained in 28/106 patients (26%). Since TBNA was performed in a general hospital by different bronchoscopists, this procedure is useful in the workup of lung cancer patients with enlarged lymph nodes.

Effect of dietary inulin supplementation on inflammation of pouch mucosa in patients with an ileal pouch-anal anastomosis.

PURPOSE: Inflammation is a constant finding in the ileal reservoir of patients with an ileal pouch-anal anastomosis and is associated with decreased fecal concentrations of the short chain fatty acid butyrate, increased fecal pH, changes in fecal flora, and increased concentrations of secondary bile acids. In healthy subjects, inulin, a dietary fiber, is fermented to short chain fatty acids and leads to a lower pH and potentially beneficial changes in fecal flora. The aim of the present study was to investigate the effect of enteral supplementation of inulin on inflammation of the ileal reservoir. METHODS: Twenty patients received 24 g of inulin or placebo daily during three weeks in a randomized, double-blind, crossover design. Stools were analyzed after each test period for pH, short chain fatty acids, microflora, and bile acids. Inflammation was assessed endoscopically, histologically, and clinically. RESULTS: Compared with placebo, three weeks of dietary supplementation with 24 g of inulin increased butyrate concentrations, lowered pH, decreased numbers of Bacteroides fragilis, and diminished concentrations of secondary bile acids in feces. This was endoscopically and histologically accompanied by a reduction of inflammation of the mucosa of the ileal reservoir. CONCLUSION: Enteral inulin supplementation leads to a decrease of inflammation-associated factors and to a reduction of inflammation of pouch mucosa.