Johan Botling

HRG Inhibits Tumor Growth and Metastasis by Inducing Macrophage Polarization and Vessel Normalization through Downregulation of PlGF

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

In situ sequencing for RNA analysis in preserved tissue and cells

Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.

Biobanking of fresh frozen tissue : RNA is stable in nonfixed surgical specimens

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6–16 h. Gene expression by real-time PCR of cfos, HIF1α, Bcl2, PCNA, TGFβ1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2–3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

A Comprehensive Analysis of Human Gene Expression Profiles Identifies Stromal Immunoglobulin κ C as a Compatible Prognostic Marker in Human Solid Tumors

Purpose: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. Experimental Design: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell–derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non–small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. Results: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. Conclusion: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining. Clin Cancer Res; 18(9); 2695–703. ©2012 AACR.

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.

Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.MethodsQuantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.ResultsAberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.ConclusionsQuantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

Background Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined. Materials and Methods The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers. Results G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005). Discussion Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer

Ductal carcinoma in situ (DCIS) is a non‐invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT‐PCR was used for validation. All DCIS and invasive samples could be classified into the “intrinsic” molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER‐status and HER2‐status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT‐PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re‐organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a womans risk of progression, and lead to improved management that would avoid the current over‐ and under‐treatment of patients.