BMC Clinical Pathology

As the major subfamily of receptor tyrosine, erythropoietin-producing hepatocellular (Eph) receptor has been related to progression and prognosis in different types of tumors. However, the role and mechanism of EPHA3 in gastric cancer is still not well understood. Specimen were collected from 202 patients who underwent gastric resection for gastric adenocarcinoma. The expression of EphA3 was studied using immunohistochemistry. We analyzed the clinicopathological factors and prognostic relevance of EphA3 expression in gastric cancer. High expression of EphA3 was associated with male predominance (p = 0.031), differentiated histology (p < 0.001), depth of tumor (p = 0.002), lymph node metastasis (p = 0.001), distant metastasis (p = 0.021), liver metastasis (p = 0.024), advanced stage (p < 0.001), and high HER2 expression (p = 0.017). Relapse-free survival (RFS) was significantly worse in patients with high expression of EphA3 than in those with low expression of EphA3 (p = 0.014). Multivariate analysis for RFS showed that depth of tumor [hazard ratio (HR) 9.333, 95% confidence interval (CI) 2.183–39.911, p = 0.003] and lymph node metastasis [hazard ratio (HR) 5.734, 95% confidence interval (CI) 2.349–13.997, p < 0.001] were independent prognostic factors. These findings suggest that high expression EphA3 may participate in metastasis and worse survival.

Chromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques. Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory. The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit. The concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.

Angioimmunoblastic T-cell lymphoma is one of the most common types of peripheral T-cell lymphomas, usually presenting at an older age with an aggressive clinical course. Its characteristic morphological presentation and follicular helper T-cell phenotype help to distinguish it from other T-cell lymphomas. We recently encountered the unique case of a 63-year old patient with relapsed tumour-cell rich angioimmunoblastic T-cell lymphoma, presenting with a “classical” phenotype and, in addition, an acquired, strong, aberrant expression of CD20. “Lineage infidelity” of phenotypic markers is a well-documented phenomenon in lymphomas and leukemias, a circumstance currently still poorly understood and with the potential to bring about erroneous interpretations, causing diagnostic havoc. This case represents one of the few documented angioimmunoblastic T-cell lymphomas with strong CD20 expression. Of interest, CD20 expression was only detected in the recurrent lymphoma and not upon initial diagnosis. The clinical importance of this finding lies in the potential for treatment with an anti-CD20 antibody, for instance Rituximab, in addition to standard chemotherapy protocols for angioimmunoblastic T-cell lymphoma. Diagnostic work-up of lymphomas to determine their lineage should therefore consider morphology, pheno- as well as genotypic characteristics, where appropriate, and in particular signs of progression and change in marker profile in relapsed cases e.g. acquisition of “non-lineage” markers such as CD20 in T-cell lymphoma.

Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample.

Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing.

In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples.

MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.

Lysine-specific demethylase1 (LSD1) is a nuclear protein which belongs to the aminooxidase-enzymes playing an important role in controlling gene expression. It has also been found highly expressed in several human malignancies including breast carcinoma. Our aim was to detect LSD1 expression also in pre-invasive neoplasias of the breast. In the current study we therefore analysed LSD1 protein expression in ductal carcinoma in situ (DCIS) in comparison to invasive ductal breast cancer (IDC). Using immunohistochemistry we systematically analysed LSD1 expression in low grade DCIS (n = 27), intermediate grade DCIS (n = 30), high grade DCIS (n = 31) and in invasive ductal breast cancer (n = 32). SPSS version 18.0 was used for statistical analysis. LSD1 was differentially expressed in DCIS and invasive ductal breast cancer. Interestingly, LSD1 was significantly overexpressed in high grade DCIS versus low grade DCIS. Differences in LSD1 expression levels were also statistically significant between low/intermediate DCIS and invasive ductal breast carcinoma. LSD1 is also expressed in pre-invasive neoplasias of the breast. Additionally, there is a gradual increase of LSD1 expression within tumour progression from pre-invasive DCIS to invasive ductal breast carcinoma. Therefore upregulation of LSD1 may be an early tumour promoting event.

Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test. The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

Lumbar puncture with quantification of leukocytes and differential count of cellular subsets in the cerebrospinal fluid is a standard procedure in cases of suspected neuroinfectious conditions. However, a number of non-infectious causes may result in a low leukocyte number (0–1000 cells/ml). We wanted to assess the diagnostic diversity of unselected adult patients with pleocytosis in the cerebrospinal fluid. The study is based on data from cerebrospinal fluid (CSF) analyses of all adult patients (15 years or older) admitted to a large university hospital in Denmark during a two-year period (2008–2009). Data from the local patient administrative system supplied with data from patient charts were combined with laboratory data. A total of 5390 cerebrospinal fluid samples from 3290 patients were included. Pleocytosis >5 leucocytes/μl was found in samples from 262 patients of which 106 (40.5%) were caused by infection of the central nervous system (CNS), 20 (7.6%) by infection outside CNS, 79 (30.2%) due to non-infectious neurological diseases, 23 (8.8%) by malignancy, and 34 (13.0%) caused by other conditions. Significantly higher mean CSF leukocytes was found in patients suffering from CNS infection (mean 1135 cells/μl, p-value <0.0001). CNS infection, non-infectious neurological disease, malignancy, and infection outside CNS can cause pleocytosis of the cerebrospinal fluid. Leukocyte counts above 100/μl is mainly caused by CNS infection, whereas the number of differential diagnoses is higher if the CSF leukocyte counts is below 50/μl. These conditions are most commonly caused by non-infectious neurological diseases including seizures.

EGFR and HER2 overexpression has been reported to play important roles in colorectal cancer (CRC) development and metastasis. Ovarian metastasis is rare yet is one of the most malignant metastases of CRC, but very few studies have focused on its biological features. This study aimed to investigate the expression of EGFR and HER2 in ovarian metastases of CRC and to reveal their clinical significance. The expression of HER2 and EGFR in both primary tumours and ovarian metastases was analysed by immunohistochemistry (IHC) in 31 CRC patients with ovarian metastases as well as in the primary tumours of 26 CRC patients with non-ovarian metastases. The overall survival time was calculated with a Kaplan-Meier survival curve and compared with a log-rank test. HER2 positivity in primary tumours was significantly higher in patients with ovarian metastases than in those with non-ovarian metastases (54.5% vs. 36.4%, P < 0.05). The EGFR-positive rate in primary lesions was not significantly different between patients with ovarian metastases and those with non-ovarian metastases (63.6% vs. 58.3%, P > 0.05). HER2 expression was not correlated with age, primary tumour site, tumour differentiation, tumour diameter or vascular cancer embolus (P > 0.05). The positive rates of HER2 and EGFR in ovarian metastases were 44.8 and 69.0%, respectively. HER2 expression in ovarian metastases was correlated with peritoneal metastasis and bilateral ovarian metastasis (P < 0.05) but not with age, synchronous or metachronous ovarian metastases and the primary tumour site (P > 0.05). There was no significant correlation between EGFR expression and the clinicopathological features in ovarian metastases (P > 0.05). CRC patients with HER2-positive ovarian metastases showed a shortened overall survival time compared to that of CRC patients with HER2-negative metastases (17.0 ± 5.2 vs. 32.0 ± 8.3 months). Our studies revealed that EGFR and HER2 are highly expressed in the primary tumours and metastases of CRC patients with ovarian metastases. HER2 positivity may be a negative prognostic predictor in patients with ovarian metastases.