Detecting EGFR alterations in clinical specimens-pitfalls and necessities.

We investigated the epidermal growth factor receptor (EGFR) status in early stage lung cancer in Southern Sweden, a population for which there are no previous reports on the EGFR mutation frequency. Three hundred fifty small cell lung cancers, adenocarcinomas (AC), squamous cell carcinomas (SqCC), and large cell carcinomas were analyzed using a combination of techniques for the analysis of protein expression, gene copy numbers, and mutations. Immunohistochemical (IHC) staining with antibodies for the EGFR mutations L858R and del E746-A750 revealed intratumoral heterogeneity and several discrepant cases when compared to mutation-specific polymerase chain reaction (PCR)-based analysis. The frequencies of these two mutations, when considering IHC staining with mutation-specific antibodies in a cohort of 298 cases and subsequent confirmation by PCR, were 10 % in AC and <2 % in SqCC. Furthermore, screening by sequencing of EGFR in a cohort of 52 lung AC and squamous carcinomas demonstrated a more diverse mutation spectrum, not covered by the mutation-specific antibodies. High expression of total EGFR protein was correlated to high gene copy numbers but did not reflect the mutational status of the tumors. We believe that the mutation spectra in a Southern Swedish population is too diverse to be covered by the mutation-specific antibodies, and we also raise some other issues regarding the use of the mutation-specific antibodies, for example concerning heterogeneous expression of the mutated protein, optimal antibody dilution, and discrepancies between staining results and PCR.

Epidermal growth factor receptor and vascular endothelial growth factor receptor 2 are specific biomarkers in triple-negative breast cancer. Results from a controlled randomized trial with long-term follow-up.

Triple-negative breast cancer (TNB) has poor prognosis and moreover patients with TNB do not benefit from established targeted drugs with endocrine therapy or trastuzumab. The aim of the study was to analyze the prevalence of candidate biomarkers in tumors from patients with TNB. Tissue microarrays were prepared from primary tumors from premenopausal breast cancer patients (500/564) randomized to adjuvant tamoxifen or no adjuvant treatment. Immunohistochemical (IHC) staining included ER, PR, HER2, epidermal receptor growth factor (EGFR), vascular endothelial growth factor A (VEGF-A), and vascular endothelial growth factor receptor 2 (VEGFR2). EGFR and HER2 gene copy number was defined by fluorescence in situ hybridization (FISH). All patients were included in the descriptive analysis, but only untreated patients in the survival analysis. TNB was diagnosed in 96 patients and correlated significantly to low age, Nottingham histological grade (NHG) III, high Ki67-index, T2 tumors, node negativity, EGFR positivity, increased EGFR gene copy number and high VEGFR2 expression. TNB was an independent prognostic factor for decreased 5-year breast cancer specific survival (BCSS) (HR 2.0 (95% CI 1.1-3.6), P = 0.01), but not for 10-year BCSS. High VEGFR2 expression was significantly correlated to decreased BCSS in TNB patients. TNB was associated with decreased BCSS and clinicopathological characteristics of an aggressive tumor type. High VEGFR2 expression, EGFR expression, and EGFR gene copy number were significantly correlated to TNB, supporting their role as putative candidate biomarkers for selection of targeted therapy in TNB.

Reproducibility of human epidermal growth factor receptor 2 analysis in primary breast cancer: a national survey performed at pathology departments in Sweden.

BACKGROUND: HER2 is a treatment predictive factor for the effect of trastuzumab and associated with poor prognosis in breast cancer. The analysis of HER2 must be performed with good quality, with regard to both the immunohistochemical (IHC) and in situ hybridization (ISH) analysis. MATERIAL AND METHODS: A tissue microarray (TMA) including 11 breast cancer samples was sent twice (once in 2005 and again in 2006) to 24 pathology departments in Sweden. A questionnaire was also sent to the departments in 2006. RESULTS: With IHC, all departments reported the same results (0/1+ vs. 2+ vs. 3 + ) for three (2005) and six samples (2006). The mean kappa-value increased from 0.67 to 0.77, indicating a good reproducibility at both occasions. With fluorescence-ISH (FISH), the 11 departments using this technique reported the same results (amplified vs. normal) for nine (2005) and ten samples (2006). The mean kappa-value showed very good reproducibility both 2005 and 2006 (0.92 and 0.96, respectively). Based on the answers from the participating departments, the questionnaire revealed that 31% of primary breast cancer diagnosed in 2006 (n = 5 043) were 2 + /3+. FISH analysis of 2+ confirmed 12% of the samples to be amplified. The corresponding figure for 3 + was 90%. In total, 14.3% of the samples were HER2 positive (2+ and amplified, or 3 + ). DISCUSSION: The results obtained in this study indicate that the reproducibility for HER2 analysis is good (IHC) and very good (FISH) between the pathology departments in Sweden using TMA-based tumor samples. In 2006, 14.3% of invasive breast cancers were HER2 positive.

Dietary supplementation with carbonate increases expression of ornithine decarboxylase and proliferation in gastric mucosa in a rat model of gastric cancer.

Dietary factors play essential roles in gastric carcinogenesis. We recently found that dietary supplementation with NaHCO(3) significantly increased the development of gastric cancer in a rat gastric stump model. Here, we analysed nontransformed gastric mucosa for expression of the cancer-related proteins cyclooxygenase-2 (COX-2) and ornithine decarboxylase (ODC), and we examined the relationship between expression levels of those proteins and mucosal proliferation. Research has shown that COX-2 is upregulated in gastric mucosal inflammation and is strongly associated with gastrointestinal cancer. ODC is the key enzyme in polyamine synthesis and a regulator of cell proliferation. We performed gastric resections on 48 Wistar rats to induce spontaneous gastric cancer; half of these animals were given a normal diet, and the other half received a diet supplemented with NaHCO(3). Twenty-four unoperated rats served as a control group. The surgical procedure per se led to a significant rise in mucosal expression of COX-2 and an associated increase in cell proliferation. However, the COX-2 level in gastric mucosa was not further affected by dietary supplementation of carbonate. Interestingly, nontransformed gastric mucosa in the operated rats receiving a carbonate-supplemented diet showed a pronounced increase in ODC expression that was strongly correlated with a further enhanced cell proliferation. These results indicate that carbonate ions, which represent a major constituent of intestinal reflux into the stomach, increase the expression of ODC and thereby enhance cell proliferation in nontransformed mucosa, and consequently elevate the risk of gastric cancer.

Chronic intestinal pseudo-obstruction due to buserelin-induced formation of anti-GnRH antibodies.

BACKGROUND & AIMS: A 30-year-old woman, treated with buserelin, an analogue of gonadotropin-releasing hormone (GnRH) (also called luteinizing hormone-releasing hormone, LH-RH), developed chronic intestinal pseudo-obstruction (CIPO). The sudden onset of this disease in a previously healthy woman perplexed us. CIPO refers to a gastrointestinal disorder that can have a variety of causes, such as drugs, among others. Thus, we wanted to examine whether in this patient the development of CIPO is related to the treatment with buserelin. METHODS: The patient was examined using esophagogastroduodenoscopy, esophageal, and antroduodenojejunal manometry, gastric emptying tests, and histologic analyses and immunohistochemistry on full-thickness biopsies including staining with anti-GnRH antibody. Plasma samples were examined by the standard serologic analyses and specifically for the occurrence of anti-GnRH antibodies by enzyme-linked immunosorbent assay methods. RESULTS: CIPO was diagnosed based on symptoms (abdominal pain, vomiting, and constipation), and the results of the clinical examinations, such as signs of esophageal aperistalsis, delayed gastric emptying, and small intestinal bursts. Histologic examination revealed a decreased number of myenteric neurons as well as increased neuronal degeneration and an abnormal immune profile. There was a loss of GnRH-containing neurons. The patient had high plasma titers of anti-GnRH antibodies, which occurred on the occasions of the treatment with buserelin. CONCLUSIONS: Our findings suggest that the patient has developed CIPO due to buserelin-induced formation of anti-GnRH antibodies destroying GnRH-producing neurons of the myenteric plexus.

[HER2 testing for breast cancer. Swedish laboratories can now offer a quality assured analysis of growth factor]. / HER2-testning för bröstcancer. Kvalitetssäikrad analys av tillväixtfaktor erbjuds nu på svenska laboratorier.

Between 20 and 30 per cent of all breast cancers show an overexpression of the growth factor HER2 and this implies a worse prognosis. Metastasising breast cancer that overexpress HER2 can effectively be treated with trastuzumab (Herceptin). In order to determine whether this treatment is indicated, HER2 expression must be quantified with immunohistochemical or molecular biological methods. Several factors may influence the laboratory analysis of HER2, such as choice of antibodies and probes, the reliability of the test performance itself and the performance of the pathologist(s) interpreting the test. National guidelines for HER2 testing have been published in several countries. In this article a series of quality assurance projects leading to the formulation of Swedish guidelines for HER2 testing is described. These projects have demonstrated that several Swedish pathology laboratories can reliably perform and interpret analysis of HER2 status today.