Michael Bergqvist

Biomarker Discovery in Non–Small Cell Lung Cancer: Integrating Gene Expression Profiling, Meta-analysis, and Tissue Microarray Validation

Purpose: Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multigene signatures in clinical practice is unclear, and the biologic importance of individual genes is difficult to assess, as the published signatures virtually do not overlap. Experimental Design: Here, we describe a novel single institute cohort, including 196 non–small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data were used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level P < 0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n = 860). Results: The meta-analysis revealed 14 genes that were significantly associated with survival (P < 0.001) with a false discovery rate <1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from 2 independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup. Conclusions: Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics. Clin Cancer Res; 19(1); 194–204. ©2012 AACR.

Elevated preoperative serum levels of angiogenic cytokines correlate to larger primary tumours and poorer survival in non-small cell lung cancer patients

We have analysed the predictive and prognostic information in preoperatively collected serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients clinically evaluated as operable non-small cell lung cancer (NSCLC). Fifty-eight patients with operable NSCLC were included. VEGF and bFGF levels in serum were analysed using enzyme linked immunosorbent assays (Quantikine human VEGF and Quantikine HS human FGF basic, R&D Systems). Univariate analysis demonstrated that tumour volume, platelet counts, VEGF and bFGF were significant prognostic factors. However, only bFGF remained significant in the multivariate analysis (P=0.014). Significant correlations were demonstrated between VEGF levels and tumour volume (r=0.33; P=0.012) and platelet count (r=0.43; P=0.001). bFGF levels correlated significant with recurrent disease (r=0.34; P=0.01), platelet count (r=0.53, P<0.001) and performance status (r=0.29; P=0.029). Furthermore, bFGF levels and VEGF levels correlated significantly (r=0.44; P<0.001). We conclude that elevated circulating angiogenic cytokines correlate with tumour volume, higher relapse risk and poorer survival in patients with operable non-small cell lung cancer.

Human cytogenetic biomonitoring using flow‐cytometric analysis of micronuclei in transferrin‐positive immature peripheral blood reticulocytes

We have developed a method to isolate and analyze nascent human reticulocytes in peripheral blood for the presence of micronuclei (MN). For a very short time peripheral reticulocytes show residual expression of the transferrin receptor. Using immunomagnetic separation of cells expressing the transferrin receptor, a population of immature reticulocytes (Trf‐Ret) was isolated from peripheral blood. In humans, the spleen actively removes micronucleated erythrocytes but during the short lifetime of the isolated Trf‐Ret only a fraction (less than about 20%) of the MN‐containing reticulocytes will have been eliminated. Cells were stained with the fluorescent dyes Thiazole Orange for RNA and Hoechst 33342 for DNA and analyzed by flow cytometry and fluorescence microscopy. Baseline frequencies of MN‐Trf‐Ret on a group of healthy donors were found to be 1.1% for males and 1.4% for females; however, the gender difference was not significant. The frequency of MN‐Trf‐Ret in the studied group increased with age, and was dependent on blood group. In three donors studied over 4 months, the baseline level remained stable. In cancer patients treated with radiation or chemotherapy, the frequency of MN‐Trf‐Ret increased 10‐ to 20‐fold after 1–4 days, depending on the treatment. A high correlation between flow and manual analysis of MN‐Trf‐Ret was seen. We believe the method has a high potential as a sensitive and rapid method for biological monitoring in presumed exposed groups and individuals. Environ. Mol. Mutagen. 36:22–31, 2000.

Hsp90 is expressed and represents a therapeutic target in human oesophageal cancer using the inhibitor 17-allylamino-17-demethoxygeldanamycin.

Heat shock protein 90 (Hsp90) has been demonstrated to protect oncogenic variants of signalling molecules from degradation and may consequently serve as a therapeutic target for the treatment of oesophageal cancer for which adequate therapy is often lacking. We studied the expression of Hsp90 in tumour tissues of human oesophageal cancer and the impact of Hsp90 inhibition on oesophageal cancer cell lines using the drug 17-allylamino-17-demethoxygeldanamycin (17-AAG). Quantitative immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues from patients with oesophageal cancer. In squamous cell carcinoma, a marked upregulation of Hsp90 could be noted in dysplastic epithelium and invasive cancer compared with normal epithelium. In adenocarcinoma, Hsp90 was expressed in neoplastic epithelium and also in normal non-neoplastic glands weakly. The inhibition of Hsp90 using 17-AAG led to a significant decrease in cell proliferation and viability in human oesophageal cancer cell lines. Using a clonogenic cell survival assay, Hsp90 inhibition significantly sensitised the cells for γ-photon irradiation. Heat shock protein 90 was found to be critical for proper signalling induced by both epidermal growth factor and insulin-like growth factor-1, in which the inhibition of signalling by 17-AAG correlated with the observed reduction in cell proliferation and viability. These results showed that Hsp90 was selectively expressed in oesophageal cancer tissue compared with the corresponding normal tissue, and the inhibition of Hsp90 resulted in decreased proliferation and viability as well as radiosensitisation of oesophageal cancer cells. Heat shock protein 90 represents a potential therapeutic target in the treatment of patients with oesophageal cancer, alone or in combination with radiotherapy.

The prognostic relevance of tumour-infiltrating plasma cells and immunoglobulin kappa C indicates an important role of the humoral immune response in non-small cell lung cancer

A prognostic impact of immunoglobulin kappa C (IGKC) expression has been described in cancer. We analysed the influence of B-cell and plasma cell markers, as well as IGKC expression, in non-small lung cancer (NSCLC) using immunohistochemistry on a tissue microarray. IGKC protein expression was independently associated with longer survival, with particular impact in the adenocarcinoma subgroup. Moreover, a correlation was seen with CD138+ cells, but not with CD20. CD138 expression revealed a comparable association with survival. In conclusion, IGKC expression in stroma-infiltrating plasma cells is a prognostic marker in NSCLC, supporting emerging treatment concepts that exploit the humoral immune response.

High-risk human papilloma virus (HPV) and survival in patients with esophageal carcinoma: a pilot study

BackgroundHuman papilloma virus (HPV) in patients with esophageal carcinoma has previously been studied with an average detection rate of 15%, but the role of HPV in relation to survival is less clear. In cervical cancer, lung cancer and tonsil cancer HPV viral load is a predictive factor for survival and outcome of treatment. The primary aim was to study the spectrum of high-risk HPV types in esophageal tumors. Secondary, as a pilot study we investigated the association between HPV status and the survival rates.MethodsWe compared both the presence and the viral load of high-risk HPV types 16, 18, 31, 33, 39, 45, 52, 58, and 67 in relation to clinical data from patients with esophageal carcinoma. Survival data and tumor samples were retrieved from 100 patients receiving treatment at the Department of Oncology, Uppsala Hospital, Uppsala, Sweden. The tumor samples were investigated for HPV viral load using real-time PCR.ResultsHPV 16 was detected in 16% of the patients; no other HPV type was detected. HPV 16 infection had no significant effect on survival (p = 0.72). Also, HPV 16 did not improve survival after treatment (radiotherapy or chemotherapy).ConclusionOnly HPV 16 was detected among the patients. HPV 16 in esophageal carcinoma patients did not influence survival or improve therapy response. However, given the size of the study there is a need to examine a larger cohort in order to understand in more detail the effect of high risk HPV types in esophageal carcinoma.

CD99 is a novel prognostic stromal marker in non-small cell lung cancer

The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour–stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non‐small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p = 0.037) and multivariate (p = 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p = 0.008). Furthermore, double‐staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer‐associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.

Clinical Phase I study with an Insulin-like Growth Factor-1 receptor inhibitor: experiences in patients with squamous non-small cell lung carcinoma.

Abstract Background. Inhibition of the Insulin-like Growth Factor-1 receptor (IGF-1R) has resulted in extensive anti-tumor effects. Picropdophyllin (PPP, AXL1717) is a small-molecule inhibitor of the IGF-1R without inhibition of closely related receptors including the insulin receptor and has shown extensive effects against a wide range of tumors in animals. PPP is currently tested as an orally administrated single agent treatment in an open-label combined Phase I/II clinical study in advanced cancer patients with solid tumors which progress in spite of several lines of treatment. Patients and methods. The first part (Phase IA) consisted of single day BID dosing every three weeks with consecutive dose escalations. The second part (Phase IB) consists of seven days or longer BID dosing every three weeks, dosing range being 520–700 mg BID. Non-progressing patients could continue treatment within a compassionate use setting. Results and discussion. The present report describes our experience with the four patients with progressive squamous non-small cell lung cancer (NSCLC) that have received treatment with PPP. Despite more than seven months of PPP treatment as third or fourth line treatment, the reported patients did not develop any additional metastases. Furthermore, CT scans as well as 18FDG-Positron Emission Tomography (PET) scans of the patients demonstrated large central necrotic areas, which may suggest tumor response. At the same time, the study drug is so far well tolerated. The phenomenon of necrosis in the tumors suggestive of tumor response has not been reported before in anti-IGF-1R treatment and will be subject to further studies in the present clinical trial.

HER-2, EGFR, COX-2 expression status correlated to microvessel density and survival in resected non-small cell lung cancer

The incidence of lung cancer is increasing throughout the world and is the most common cause of cancer-related death. Early detection followed by surgery has a reasonable, curative potential, but 30-50% of patients experience relapses. The immunohistochemical expressions of HER-2, EGFR and COX-2 were investigated in 53 resected non-small cell lung carcinomas and correlated to microvessel density and clinical data. HER-2, EGFR and COX-2 overexpressions were demonstrated in 15%, 30% and 40% of the tumours, respectively. In adenocarcinomas, HER-2 and COX-2 overexpression were more common, whereas in squamous cell carcinomas, EGFR overexpression was more common. COX-2 expression correlated with HER-2 expression (p = 0.002), and demonstrated a trend towards a correlation with microvessel density (p = 0.10). None of the markers alone had any impact on survival. However, HER-2+/EGFR- tumours proved to have a poor prognosis. In conclusion, adjuvant treatment with HER-2 antagonists might be a future treatment option in resected non-small cell lung cancer patients, especially when HER-2 is overexpressed without a concomitant overexpression of EGFR.

Gene copy number aberrations are associated with survival in histologic subgroups of non-small cell lung cancer.

Introduction: Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups. Methods: Short-term (<20 month; n = 53) and long-term survivors (>58 month; n = 47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh frozen tumor specimens. The samples were analyzed using high-resolution single-nucleotide polymorphism array technology to assess gene copy number variations and array-based gene expression profiling. The molecular data were combined with information on clinical parameters. Results: Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n = 50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term than in short-term survivors. In squamous cell carcinoma (n = 28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, messenger RNA expression levels of corresponding genes were increased or decreased, respectively. Conclusion: Comprehensive tumor profiling permits the integration of genomic, histologic, and clinical data. We identified gene copy number gains and losses, with corresponding changes in messenger RNA levels that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung.