Göran Elmberger

Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer

BACKGROUND Triple-negative breast cancer (TNBC) lacking expression of steroid receptors and human epidermal growth factor receptor 2, having chemotherapy as the only therapeutic option, is characterised by early relapses and poor outcome. We investigated intratumoural (i.t.) levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and survival in patients with TNBC compared with non-TNBC. PATIENTS AND METHODS VEGF levels were determined by an enzyme immunosorbent assay in a retrospective series consisting of 679 consecutive primary breast cancer patients. RESULTS Eighty-seven patients (13%) were classified as TNBC and had significantly higher VEGF levels; median value in TNBC was 8.2 pg/microg DNA compared with 2.7 pg/microg DNA in non-TNBC (P < 0.001). Patients with TNBC had statistically significant shorter recurrence-free survival [hazard ratio (HR) = 1.8; P = 0.0023], breast cancer-corrected survival (HR = 2.2; P = 0.004) and overall survival (HR = 1.8; P = 0.005) compared with non-TNBC. Patients with TNBC relapsed earlier than non-TNBC; mean time from diagnosis to first relapse was 18.8 and 30.7 months, respectively. The time between first relapse and death was also shorter in TNBC: 7.5 months versus 17.5 months in non-TNBC (P = 0.087). CONCLUSIONS Our results show that TNBC have higher i.t. VEGF levels compared with non-TNBC. Ongoing clinical trials will answer if therapy directed towards angiogenesis may be an alternative way to improve outcome in this poor prognosis group.

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.

Flow Cytometric Immunophenotyping Including Bcl-2 Detection on Fine Needle Aspirates in the Diagnosis of Reactive Lymphadenopathy and Non-Hodgkin's Lymphoma

Fine‐needle aspiration (FNA) with immunophenotyping by immunocytochemistry (IC) on cytospins has recently received increased consideration in the diagnosis of lymphoma. The aim of our study was to establish the diagnostic value of a four‐color flow cytometric (FCM) panel, including cytoplasmic Bcl‐2, in cytologic diagnosis of malignant non‐Hodgkins lymphoma (NHL) and reactive lymphoid hyperplasia (RH).

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (> or =four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression (P=0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly (P=0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers (P=0.001, P=0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

Personalized cancer medicine and the future of pathology.

In February 2011, a group of pathologists from different departments in Europe met in Zurich, Switzerland, to discuss opportunities and challenges for pathology in the era of personalized medicine. The major topics of the meeting were assessment of the role of pathology in personalized medicine, its future profile among other biomedical disciplines with an interest in personalized medicine as well as the evolution of companion diagnostics. The relevance of novel technologies for genome analysis in clinical practice was discussed. The participants recognize that there should be more initiatives taken by the pathology community in companion diagnostics and in the emerging field of next-generation sequencing and whole genome analysis. The common view of the participants was that the pathology community has to be mobilized for stronger engagement in the future of personalized medicine. Pathologists should be aware of the challenges and the analytical opportunities of the new technologies. Challenges of clinical trial design as well as insurance and reimbursement questions were addressed. The pathology community has the responsibility to lead medical colleagues into embracing this new area of genomic medicine. Without this effort, the discipline of pathology risks losing its key position in molecular tissue diagnostics.

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.

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

BackgroundIn-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.ResultsCytological 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.ConclusionThe 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.

Prostate cancer cell lines lack amplification : Overexpression of HER2

The potential overexpression of HER2 in prostate cancer cells has attended significant interest during the past few years, both as potential target for HER2 pathway focused therapy and as a mechanism involved in the progression to androgen independence. Conflicting results have been reported concerning HER2 status on clinical material, differences which generally have been attributed to methodological differences. Nevertheless, HER2 has been utilized for targeted therapy of prostate cancer in a number of preclinical studies and is still regarded as an exciting target molecule. In this study, the HER2 status of three widely used prostate cancer cell lines and corresponding xenografts has been analysed. By use of validated and FDA approved analytical staining techniques none of these cell lines or xenografts were shown to overexpress/amplify HER2, as demonstrated by immunohistochemistry and fluorescense in situ hybridization. These findings are important for the interpretation and understanding of the therapeutic effects when developing drugs targeting HER2 in prostate cancer cell lines and also emphasize the importance of using broad and validated analytical techniques.

Detection of smoothelin expression in the urinary bladder is strongly dependent on pretreatment conditions: a critical analysis with possible consequences for cancer staging

Distinguishing urinary bladder muscularis propria (MP) from muscularis mucosae (MM) is crucial in bladder cancer staging. Immunohistochemical staining for the smooth muscle-specific protein smoothelin has been reported to be a robust marker for MP. The aim of this study was to investigate how smoothelin immunostaining in the bladder varies with pretreatment techniques and if it can be used to discriminate between MM and MP. Immunohistochemistry (IHC) for smoothelin was performed on nontumoral sections from 18 cystectomy specimens using three different pretreatment protocols. The immunoreactivity of MM, MP and blood vessels was scored semiquantitatively. Staining intensity depended strongly on the different pretreatment protocols used. Heat-induced epitope retrieval (HIER) in alkaline buffer resulted in the strongest staining with a moderate or strong immunostaining of the MP in 18/18 (100%) of cases, but in 11/18 (61%), the MM was moderately or strongly stained. HIER in acidic buffer resulted in a suboptimal staining of the MP. Enzymatic pretreatment resulted in absent or weak staining. In conclusion, smoothelin IHC is strongly dependent on epitope retrieval, and smoothelin staining did not discriminate reliably between MP and MM with any of the tested pretreatment protocols.

Progressive Reactive Lymphoid Connective Tissue Disease and Development of Autoantibodies in Scavenger Receptor A5–Deficient Mice

Scavenger receptor A5 (SCARA5) is a member of the class A scavenger receptors, with most similarity to SCARA1 (SR-A) and SCARA2 (MARCO), which are primarily expressed by macrophages and dendritic cells, in which they participate in clearance of various polyanionic macromolecules, pollution particles, and pathogens. The biological role of SCARA5 has been unknown. Herein, we show that SCARA5 is an endocytotic receptor whose ligand repertoire includes the typical scavenger receptor ligands, whole bacteria, and purified Gram-negative bacterial lipopolysaccharide. In contrast to expression of SCARA1 and SCARA2 in immune cells, SCARA5 is found in a subset of fibroblast-like cells in the interstitial stroma of most organs, with additional expression in the epithelial cells of testis and choroid plexus. SCARA5-null mice develop with age lymphoid cell accumulation in many organs, in particular the lungs, and show decreased endocytotic function in fibroblasts. Furthermore, about one-third of the mice develop antinuclear antibodies. These disturbances are reminiscent of those found in many human autoimmune connective tissue disorders, which suggests that defects in fibroblast SCARA5 can underlie some forms of autoimmune disease.