Determination of phosphorylated proteins in tissue specimens requires high-quality samples collected under stringent conditions.

AIMS: For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in-vivo phosphorylation states. METHODS AND RESULTS: To investigate whether routinely collected tissue specimens retain the true phosphorylation states of a tumour's proteins, we compared protein phosphorylation states between matched tumour samples that were subjected to different ischaemic times by immunohistochemistry. The influence of formalin fixation and paraffin-embedding on phosphorylation states was investigated by comparison of matched fresh frozen and formalin-fixed paraffin-embedded surgical specimens as well as small biopsies. We show that ischaemia influences protein phosphorylation in a tumour-specific, unpredictable manner. Formalin fixation and paraffin-embedding lead to a decrease in detectable protein phosphorylation in larger surgical specimens, but not in small biopsies. CONCLUSIONS: Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large-specimen fixation.

Multiple protein analysis of formalin-fixed and paraffin-embedded tissue samples with reverse phase protein arrays.

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Effects of cold ischemia and inflammatory tumor microenvironment on detection of PI3K/AKT and MAPK pathway activation patterns in clinical cancer samples.

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.

HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing.

Trastuzumab-based therapy has been shown to confer overall survival benefit in HER2-positive patients with advanced gastric cancer in a large multicentric trial (ToGA study). Subgroup analysis identified adenocarcinomas of the stomach and gastroesophageal (GE) junction with overexpression of HER2 according to immunohistochemistry (IHC) as potential responders. Due to recent approval of trastuzumab for HER2 positive metastatic gastric and GE-junction cancer in Europe (EMEA) HER2 diagnostics is now mandatory with IHC being the primary test followed by fluorescence in situ hybridization (FISH) in IHC2+ cases. However, in order to not miss patients potentially responding to targeted therapy determination of a HER2-positive status for gastric cancer required modification of scoring as had been proposed in a pre-ToGA study. To validate this new HER2 status testing procedure in terms of inter-laboratory and inter-observer consensus for IHC scoring a series of 547 gastric cancer tissue samples on a tissue microarray (TMA) was used. In the first step, 30 representative cores were used to identify specific IHC HER2 scoring issues among eight French and German laboratories, while in the second step the full set of 547 cores was used to determine IHC HER2 intensity and area score concordance between six German pathologists. Specific issues relating to discordance were identified and recommendations formulated which proved to be effective to reliably determine HER2 status in a prospective test series of 447 diagnostic gastric cancer specimens.

Extraluminal gastrointestinal stromal tumour in the second portion of the duodenum.

The preoperative diagnosis of extraluminal gastrointestinal stromal tumours in the duodenum is difficult to establish due to their rare occurrence and the lack of pathognomonic signs. This report describes the case of a 61-year-old woman who suffered from an immunohistologically confirmed gastrointestinal stromal tumour in the second portion of the duodenum. Preoperative, abdominal, multislice computed tomography showed an extraluminal but intramural tumour located between the head of the pancreas and the duodenum. Rapid postprocessing analysis by three-dimensional, volume-rendered images revealed a strong arterial blood supply and an early draining vessel into the superior mesenteric vein during the portal-venous phase. The combination of endoscopic ultrasonography and non-invasive multislice computed tomography provided an early suggestion of gastrointestinal stromal tumour.

Acute osteoporotic and neoplastic vertebral compression fractures: fluid sign at MR imaging.

PURPOSE: To evaluate the occurrence, location, and shape of the fluid sign in acute osteoporotic and neoplastic vertebral compression fractures at magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study group comprised 87 consecutive patients with acute vertebral compression fractures due to osteoporotic (n = 52) or neoplastic (n = 35) infiltration. The MR imaging protocol included nonenhanced T1-weighted spin-echo and short inversion time inversion-recovery sequences and a 1.5-T system. Readers blinded to the outcome documented the occurrence, shape, and location of the fluid sign with consensus. The fluid sign was correlated with the cause, age, and severity of the fracture. The diagnosis was confirmed with surgery, follow-up MR imaging, clinical follow-up, or unequivocal imaging findings. Wilcoxon and chi(2) tests were used to assess significance. RESULTS: In fractured vertebral bodies, the fluid sign was adjacent to the fractured end plates and exhibited signal intensity isointense to that of cerebrospinal fluid. The fluid sign was linear (n = 16), triangular (n = 5), or focal (n = 2) and was significantly associated with osteoporotic fractures (21 [40%] of 52; P <.001). The fluid sign occurred in two (6%) of 35 neoplastic compression fractures. Histologic examination demonstrated osteonecrosis, edema, and fibrosis at the site of the fluid sign. There was a tendency toward older fractures exhibiting the fluid sign, but this relationship was not significant (P >.05). In osteoporotic fractures, the fluid sign was significantly associated with fracture severity (P <.05). CONCLUSION: The fluid sign is featured in acute vertebral compression fractures that show bone marrow edema. It can be an additional sign of osteoporosis and rarely occurs in metastatic fractures.

A comparative study of normal inspection, autofluorescence and 5-ALA-induced PPIX fluorescence for oral cancer diagnosis.

Fluorescence diagnosis aims to improve the management of oral cancer via early detection of the malignant lesions and better delimitation of the tumor margins. This paper presents a comparative study of normal inspection, combined fluorescence diagnosis (CFD) and its 2 main components, autofluorescence and 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence. Biopsy-controlled fluorescence imaging and spectral analysis were performed on a total of 85 patients with suspected or histologically proven oral carcinoma both before and after topical administration of 5-ALA (200 mg 5-ALA dissolved in 50 ml of H(2)0). Fluorescence excitation was accomplished using filtered light of a xenon short arc lamp (lambda = 375-440 nm). As for CFD, a "streetlight" contrast (red to green) was readily found between malignant and healthy tissue on the acquired images. In terms of tumor localization and delimitation properties, CFD was clearly favorable over either normal inspection or its 2 components in fluorescence imaging. The performance of CFD was found to be impeded by tumor keratinization but to be independent of either tumor staging, grading or localization. In spectral analysis, cancerous tissue showed significantly higher PPIX fluorescence intensities and lower autofluorescence intensities than normal mucosa. There is a great potential for CFD in early detection of oral neoplasms and exact delimitation of the tumors' superficial margins and an advantage over white light inspection and each of its 2 main components. The method is noninvasive, safe and easily reproducible.