A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants

<p>Abstract</p> <p>Background</p> <p>In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS) from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas) and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens. By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples.</p> <p>Results</p> <p>Cytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%. The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively). Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples.</p> <p>Conclusion</p> <p>The method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method was an effective removal of contaminants from red blood cells and plasma proteins resulting in larger proteome coverage compared to the direct lysis of frozen samples. This sample preparation method may be successfully implemented for the discovery of lung cancer biomarkers on tissue samples using mass spectrometry-based proteomics.</p

Validation of Interobserver Agreement in Lung Cancer Assessment: Hematoxylin-Eosin Diagnostic Reproducibility for Non–Small Cell Lung Cancer: The 2004 World Health Organization Classification and Therapeutically Relevant Subsets

Precise subtype diagnosis of non–small cell lung carcinoma is increasingly relevant, based on the availability of subtype-specific therapies, such as bevacizumab and pemetrexed, and based on the subtype-specific prevalence of activating epidermal growth factor receptor mutations

Sclerosing Sweat Duct-Like Carcinoma of the Tongue-A Case Report and a Review of the Literature.

In this article, we present the second case of a tongue carcinoma with distinct morphological similarities to a primary sclerosing sweat duct carcinoma of the skin. The tumor infiltrated deeply into the striated muscle and exhibited extensive peri- and intraneural growth. Neoplastic cells had mildly pleomorphic nuclei and displayed in addition to small cysts, distinct duct formation with a bilayered epithelium. Mitotic figures were scarce. Despite extensive sampling, no precursor lesion-dysplasia-was found. The immunochemical study showed positive reaction for carcinoembryonic antigen, epithelial membrane antigen, cytokeratin (CK)7, low-molecular weight CK, BerEp4, high-molecular weight CK, CK5, and CK18. p63 was positive in the peripheral parts of the neoplastic ducts with negative reaction in the luminal cells

Experiences with an International Digital Slide Based Telepathology System for Routine Sign-out between Sweden and Hungary

Digital microscopy combines the benefits of traditional optical microscopy and the advantages of computer sciences. Using digital whole slides in all areas of pathology is increasingly popular. Telepathology or long distance diagnosis is one such area. In our study we have evaluated digital slide based histopathology diagnosis in an international setting, between Sweden and Hungary. Routine cases from the Sundsvall County Hospital (Landstinget Vasternorrland) were collected. Glass slides were scanned using Pannoramic 250 Flash II. (3DHISTECH Ltd., Budapest, Hungary). During the first round of evaluation the glass slides were shipped to Hungary for primary diagnosis. Two pathologists from Hungary, reading glass slides and one pathologist from Sweden reading digital slides signed out 500 cases. Pathologists from Hungary reached the hospital information system with a secure connection. During the second round the pathologists in Hungary reevaluated 200 from the 500 cases using digital slides after three months washout period. Diagnostic accuracy was calculated and diagnostic errors was graded according to clinicopathological consequences. In 182/200 (91%) cases digital and optical diagnoses were in full agreement. Out of the remaining 18 cases, 1 (0.5%) critical error was identified. In this case the error had therapeutic and prognostic consequence and no uncertainty either because of case complexity or poor image quality was recorded by the pathologist. We think language and communication issues as well as differences in minimal data sets of pathological reports and in guidelines used in Sweden and in Hungary are factors potentially limiting the widespread use of digital slides in a teleconsultation service provided to Sweden from Hungary. We found the quality of digital slides in our study setting acceptable to reach correct primary diagnosis in routine, unselected, random cases of a small-to-medium sized pathology department in Sweden

Targeted sequencing may facilitate differential diagnostics of pulmonary tumours: a case series

Abstract Background Histopathological diagnosis is important for prognostication and choice of treatment in patients with cancer in the lung. Metastases to the lungs are common and need to be distinguished from primary lung cancer. Furthermore, cases with synchronous or metachronous primary lung cancers (although infrequent) are often handled differently than cases with lung cancer with intrapulmonary metastasis or relapse, respectively. In some cases, morphology and immunohistochemical staining is not sufficient for certain diagnosis. Methods The present study included six cases where molecular genetic analysis in form of pyrosequencing or targeted next-generation sequencing was of value for certain diagnosis of selected tumours in the lung. Results Two of the included cases were rare metastases to the lung; colorectal cancer with IHC profile consistent with primary lung cancer and malignant adenomyoepithelioma of the breast, respectively, where molecular genetic analysis was of aid for proving the relationship to the primary tumour. The other four cases were multiple lung adenocarcinomas where molecular genetic analysis was of aid to distinguish between intrapulmonary metastasis and synchronous tumour. Conclusions Comparison of molecular genetic profile may be an important tool for determination of relationship between tumours in some situations and should always be considered in unclear cases. Further studies on concordance and discordance of molecular genetic profiles between spatially or temporally different tumours with common origin may be helpful for improved diagnostics of pulmonary tumours

Highly reproducible results of breast cancer biomarkers when analysed in accordance with national guidelines - a Swedish survey with central re-assessment.

Biomarkers are crucial for decisions regarding adjuvant therapy in primary breast cancer, and their correct assessment is therefore of the utmost importance

EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations

In non-small cell lung cancer, epidermal growth factor receptor gene mutations and anaplastic lymphoma kinase (ALK) gene rearrangements have a major impact upon the level of response to treatment with specific tyrosine kinase inhibitors. This review describes the molecular basis of ALK inhibition, summarizes current data on the effectiveness and safety of ALK inhibition therapy, describes the different testing methodologies with their advantages and disadvantages, provides a suggested testing algorithm and puts forward a proposal for an external quality assessment program in ALK testing