Annette Fisseler-Eckhoff

Survival after trimodality therapy for malignant pleural mesothelioma: Radical Pleurectomy, chemotherapy with Cisplatin/Pemetrexed and radiotherapy

INTRODUCTION The role of surgery in the management of malignant pleural mesothelioma (MPM) is controversial and there are no established guidelines. We describe the feasibility and long-term outcomes associated with Radical Pleurectomy (RP) as surgical therapy modality in a standardized trimodality therapy concept of MPM. METHODS From November 2002 to October 2007, 35 out of 102 consecutive patients with MPM were enrolled in our prospective database. They underwent trimodality therapy, including RP followed by 4 cycles of chemotherapy with Cisplatin (75 mg/m(2))/Pemetrexed (500 mg/m(2)) and radiotherapy 4-6 weeks after operation. RESULTS Median age was 65 years. Nineteen patients were in advanced stages III and IV (54.3%). Tumor histology was epithelial in 27 patients (77.1%). Macroscopic complete resection could be achieved in 18 patients (51.4%). Surgical morbidity/mortality and trimodality treatment-related mortality were 20.0%, 2.9% and 5.8%, respectively. Thirty-three patients completed the trimodality therapy. Median follow-up was 21.7 months. Overall median survival was 30.0 months. One-, 2-, and 3-year-survival were 69%, 50% and 31%, respectively. Advanced stages III/IV (p=0.06), macroscopic incomplete resections (p=0.001), non-epithelial histology (p=0.55) and nodal metastases (p=0.19) were associated with poorer survival. CONCLUSIONS The trimodality therapy concept with RP demonstrates promising results in terms of long-term survival, morbidity and mortality. We propose that a surgical philosophy of limiting the procedure related morbidity while achieving comparable cytoreductive results allows patients to maintain physiological reserve to be eligible for multimodality treatment options in the long-term. The observed and theoretical benefits of this trimodality treatment approach warrant confirmation in larger RCT.

Pattern and clinical predictors of lymph node metastases in epithelial ovarian cancer

Para-aortic lymphadenectomy is part of staging in early epithelial ovarian cancer (EOC) and could be part of therapy in advanced EOC. However, only a minority of patients receive therapy according to guidelines or have attendance to a specialized unit. We analyzed pattern of lymphatic spread of EOC and evaluated if clinical factors and intraoperative findings reliably could predict lymph node involvement, in order to evaluate if patients could be identified in whom lymphadenectomy could be omitted and who should not be referred to a center with capacity of performing extensive gynecological operations. Retrospective analysis was carried out of all patients with EOC who had systematic pelvic and para-aortic lymphadenectomy during primary cytoreductive surgery. One hundred ninety-five patients underwent systematic pelvic and para-aortic lymphadenectomy. Histologic lymph node metastases were found in 53%. The highest frequency was found in the upper left para-aortic region (32% of all patients) and between vena cava inferior and abdominal aorta (36%). Neither intraoperative clinical diagnosis nor frozen section of pelvic nodes could reliably predict para-aortic lymph node metastasis. The pathologic diagnosis of the pelvic nodes, if used as diagnostic tool for para-aortic lymph nodes, showed a sensitivity of only 50% in ovarian cancer confined to the pelvis and 73% in more advanced disease. We could not detect any intraoperative tool that could reliably predict pathologic status of para-aortic lymph nodes. Systematic pelvic and para-aortic lymphadenectomy remains part of staging in EOC. Patients with EOC should be offered the opportunity to receive state-of-the-art treatment including surgery

NO Signaling Confers Cytoprotectivity through the Survivin Network in Ovarian Carcinomas

Despite considerable success in the treatment of epithelial ovarian cancer (EOC), therapy resistance counteracts improvement of long-term survival. The dual role of survivin as an apoptosis inhibitor and mitotic regulator has been associated with disease outcome. However, the molecular mechanisms involved in the deregulated expression in EOC of survivin need further investigation. Here, we show that high amounts of the nitric oxide (NO) donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or strong overexpression of the inducible nitric oxide synthase (iNOS) suppressed survivin levels via the p38MAPK pathway and triggered apoptosis in ovarian cancer cell lines (OCC). Importantly, low NO concentrations conferred resistance against carboplatin/paclitaxel-induced apoptosis. Cytoprotection was mediated by survivin because we observed its up-regulation subsequent to low SNAP/SNP doses or ectopic expression of low amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling. Induction of survivin involves activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which was antagonized by the PI3K-inhibitor, LY294002. Interestingly, application of the iNOS-inhibitor 1400W together with RNAi-mediated survivin down-regulation cooperatively enhanced drug-induced apoptosis in OCCs. The iNOS/survivin interdependencies seem to be also of clinical relevance because immunohistochemistry revealed that low iNOS levels correlate with survivin expression (P < 0.01) in carboplatin/paclitaxel-treated EOC patients with minimal postoperative residual tumor (n = 54). Also, iNOS and survivin expression were associated with increased risk for disease progression. Our study uncovers a novel molecular mechanism of how NO signaling may contribute to therapy resistance in EOC by modulating survivin expression. Pharmacogenetic iNOS/survivin-targeting strategies may hence be pursued to complement current treatment modalities in EOC.

Proficiency testing of immunohistochemical biomarker assays in breast cancer

Steroid hormone receptor expression and HER2 status have become an integral part of histopathologic characterization of breast cancer and corresponding biomarker assays have gained important prognostic and predictive impact. Because testing inaccuracy could provide a major hazard to modern breast cancer therapy, a laboratory proficiency testing program has been implemented in Germany using tissue microarrays (TMAs). In four consecutive annual trials with 142 laboratories participating on average per trial, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2) were determined immunohistochemically by participating laboratories followed by central review of all immunostains. Performance strongly depended on the ambiguity of expression of the target molecule in the test samples. In clearly positive (Allred score 7–8; Her2 3+) or negative tissue samples, the majority of participants (86%) achieved concordance rates exceeding 85%. By contrast, low expression of ER or PR (Allred score 3–4) as well as Her2 status 2+ led to considerable lower concordance rates ranging from 41% (Her2 2+) to 75% (PR). Poor reproducibility was predominantly due to inadequate laboratory performance whereas interobserver agreement (weighted kappa statistics) usually was high (>0.81). Laboratories that participated in more than one of the four subsequent trials (n = 110) showed a highly significant improvement of performance. In conclusion, a TMA-based proficiency testing of biomarkers in breast cancer has been implemented in Germany over a 5-year period and revealed reliable assessment of unambiguously positive and negative test samples. Low-expressing tumor samples with regard to steroid hormone receptor expression and Her2 status 2+ led to inaccurate evaluations by up to 59% of participants. Regularly participating laboratories showed a significant improvement of performance.

Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids

Evaluation of proliferative activity is a cornerstone in the classification of endocrine tumors; in pulmonary carcinoids, the mitotic count delineates typical carcinoid (TC) from atypical carcinoid (AC). Data on the reproducibility of manual mitotic counting and other methods of proliferation index evaluation in this tumor entity are sparse. Nine experienced pulmonary pathologists evaluated 20 carcinoid tumors for mitotic count (hematoxylin and eosin) and Ki-67 index. In addition, Ki-67 index was automatically evaluated with a software-based algorithm. Results were compared with respect to correlation coefficients (CC) and kappa values for clinically relevant grouping algorithms. Evaluation of mitotic activity resulted in a low interobserver agreement with a median CC of 0.196 and a median kappa of 0.213 for the delineation of TC from AC. The median CC for hotspot (0.658) and overall (0.746) Ki-67 evaluation was considerably higher. However, kappa values for grouped comparisons of overall Ki-67 were only fair (median 0.323). The agreement of manual and automated Ki-67 evaluation was good (median CC 0.851, median kappa 0.805) and was further increased when more than one participant evaluated a given case. Ki-67 staining clearly outperforms mitotic count with respect to interobserver agreement in pulmonary carcinoids, with the latter having an unacceptable low performance status. Manual evaluation of Ki-67 is reliable, and consistency further increases with more than one evaluator per case. Although the prognostic value needs further validation, Ki-67 might perspectively be considered a helpful diagnostic parameter to optimize the separation of TC from AC.

Environmental Isocyanate-Induced Asthma: Morphologic and Pathogenetic Aspects of an Increasing Occupational Disease

Occupational diseases affect more and more people every year. According to the International Labour Organization (ILO), in 2000 an estimated amount of at least 160 million people became ill as a result of occupational-related hazards or injuries. Globally, occupational deaths, diseases and injuries account for an estimated loss of 4% of the Gross Domestic Product. Important substances that are related to occupational diseases are isocyanates and their products. These substances, which are used in a lot of different industrial processes, are not only toxic and irritant, but also allergenic. Although the exposure to higher concentrations could be monitored and restricted by technical means, very low concentrations are difficult to monitor and may, over time, lead to allergic reactions in some workers, ending in an occupational disease. In order to prevent the people from sickening, the mechanisms underlying the disease, by patho-physiological and genetical means, have to be known and understood so that high risk groups and early signs in the development of an allergic reaction could be detected before the exposure to isocyanates leads to an occupational disease. Therefore, this paper reviews the so far known facts concerning the patho-physiologic appearance and mechanisms of isocyanate-associated toxic reactions and possible genetic involvement that might trigger the allergic reactions.

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): results of a multi-centre ALK-testing.

BACKGROUND The reliable identification of non-small cell lung cancers (NSCLC) with chromosomal breaks in the gene of the anaplastic lymphoma kinase (ALK) is crucial for the induction of therapy with ALK-inhibitors. In order to ensure a reliable detection of ALK-breaks by means of fluorescence in situ hybridization (FISH) testing, round robin tests are essential. In preparation of a nation (German)-wide round robin test we initiated a pre-testing phase involving 8 experts in FISH-diagnostics to identify NSCLC cases (n = 10) with a pre-tested ALK-status. In addition, ALK immunohistochemistry (IHC) was performed to assess ALK protein expression. MATERIAL AND METHODS Sections derived from a tissue microarray, each consisting of 3 cores from 10 NSCLC cases, were independently tested for ALK protein expression by IHC and genomic ALK-breaks by FISH involving 8 institutes of pathology. Based on a pre-screening, 5 cases were identified to be clearly ALK-break negative, whereas the remaining 5 cases were ALK-break positive including one case with low percentage (20%) of positive cells. The latter had been additionally tested by RT-PCR. RESULTS The 5 unequivocal ALK-break negative NSCLC were almost consistently scored negative by means of FISH and IHC by all 8 experts. Interestingly, 4 of the 5 cases with pre-defined ALK-breaks revealed homogenous FISH results whereas IHC for the detection of ALK protein expression showed heterogeneous results. The remaining case (low number of ALK-break positive cells) was scored negative by 3 experts and positive by the other 5. RT-PCR revealed the expression of an EML4-ALK fusion gene variant 1. CONCLUSION ALK-break negative NSCLC cases revealed concordant homogeneous results by means of FISH and IHC (score 0-1) by all 8 experts. Discordant FISH results were raised in one ALK-break positive case with a low number of affected tumor cells. The remaining 4 ALK-break positive cases revealed concordant FISH data whereas the ALK-IHC revealed very diverse results. The cases with concordant FISH results provide an excellent basis for round robin ALK-FISH testing. As long as standardized ALK-IHC protocols are missing, ALK protein expression cannot by regarded as the method of choice for identification of patients eligible for treatment with ALK inhibitors.

Multicenter Immunohistochemical ALK-Testing of Non–Small-Cell Lung Cancer Shows High Concordance after Harmonization of Techniques and Interpretation Criteria

Introduction: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non–small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories. Methods: After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-“borderline” character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed. Results: All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-“borderline” samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting. Conclusions: This so-called “ALK-Harmonization-Study” shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.

Expression of |[alpha]|-methylacyl coenzyme A racemase in the dysplasia carcinoma sequence associated with Barrett's esophagus

Two different studies demonstrated α-methylacyl coenzyme A racemase (AMACR) to be a highly specific marker in Barretts neoplastic lesions. Reactive atypia was positive in 3/30 cases in these studies. We present a retrospective study of early Barretts adenocarcinoma treated with surgery (2000–2005, n=29; M:F=5:1, median age 67 years). We analyzed the role of AMACR expression in reactive and neoplastic lesions associated with the disease of 77 different specimens (60 biopsy and 17 surgical specimens) of these patients. In our cohort, 70% of cases demonstrated infiltration of the submucosa, 38% were poorly differentiated, and/or 31% demonstrated lymph vessel infiltration. We used a multi-tissue array, with reactive and neoplastic samples for each patient to analyze the immunoreactivity of AMACR. Barretts epithelium that was negative for dysplasia and columnar epithelial cell changes indefinite for dysplasia (n=30) did not demonstrate AMACR immunoreactivity. AMACR immunoreactivity was demonstrated in 27% (8/30) of cases of Barretts epithelium with columnar epithelial cell changes indefinite for dysplasia. Altogether 91% of cases with low-grade dysplasia were AMACR-positive and 96% of cases with high-grade dysplasia and early Barretts adenocarcinoma were positive for AMACR. In summary, the sensitivity of AMACR expression in low-grade dysplasia and subsequent early Barretts adenocarcinoma was significantly higher in our study compared with previous data. This might be a new diagnostic marker for dysplasia carcinoma sequence in Barretts low-grade neoplastic lesions. Further studies are required to investigate this point with well-defined controls having at least 5-years follow-up.

Prognostic risk factors of early esophageal adenocarcinomas.

Objective:To define prognostic risk factors in patients with early adenocarcinomas of the esophagus (eACEs) who were treated by esophagectomy. Background:Although endoscopic resection (ER) is more accepted for eACEs limited to the mucosa, the reported prevalence of lymph node metastases once the tumor infiltrates the submucosa seems to necessitate surgery in these cases. Methods:We analyzed the results of 168 patients who had an esophageal resection because of an eACE. On the basis of specimen histologies and clinical follow-up (median, 64 months), we investigated the influence of lymph node metastases (N+), tumor infiltration depth, tumor differentiation (G1–3), and lymphatic or venous infiltration (L+ or V+) on overall and tumor-specific survival and recurrence rates. Results:The 5-year survival rate was 79%. Lymph node infiltration was the only prognostic factor for the overall survival [hazard ratio (HR), 2.856; 1.314–6.207; P = 0.008], tumor-specific survival (HR, 8.336; 2.734–25.418; P < 0.001), and tumor recurrence (HR, 8.031; 3.041–21.206; P < 0.001) that was consistently present in all multivariate hazard Cox regression analyses. A total of 47% of the patients who had an N+ status developed tumor recurrences compared with 5.2% of those who had no lymph node involvement (P = <0.001). We found a significant correlation between N+ status and increasing depth of tumor infiltration (P = 0.004), lymphatic vessel infiltration (P = 0.002), tumor differentiation (G1 + G2 vs G3; P = 0.014) and vascular infiltration (P = 0.01). Conclusions:Lymph node status is the only independent risk factor for survival and recurrence rates. Tumor infiltration depth correlates with the rate of the lymph node metastases, but a clear watershed between deep mucosal and submucosal infiltration does not exist. As a consequence, careful staging procedures, including diagnostic ER, are mandatory to determine which patients can be treated by ER and which require an esophagectomy.