Verena Tischler

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.

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

BackgroundSeveral stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.MethodsWe depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).ResultsGene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.ConclusionsData presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.

A Role for ADAM12 in Breast Tumor Progression and Stromal Cell Apoptosis

As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.

TTF1 expression in non‐small cell lung carcinoma: association with TTF1 gene amplification and improved survival

Acquired chromosomal aberrations play an important role in tumour development and progression. Such genetic alterations occur in a significant proportion of non‐small cell lung carcinomas (NSCLCs) and include amplification of 14q13.3, which contains the TTF1 gene. We asked whether TTF1 amplification is associated with increased TTF1 protein expression in NSCLCs, and whether TTF1 is associated with clinicopathological features, including patient survival. We used a FISH assay and quantitative immunohistochemical staining to interrogate a population‐based cohort of 538 NSCLCs from Swiss patients for TTF1 amplification and protein expression. We found TTF1 amplification in ∼13% of adenocarcinomas (ACs) and in ∼9% of squamous cell carcinomas (SCCs) and TTF1 amplification was associated with increased TTF1 protein expression. High‐level TTF1 expression was significantly associated with smaller tumour size, female gender and longer overall survival only among ACs (median survival 82 versus 28 months; p = 0.002). On multivariate analysis, high TTF1 expression was an independent predictor of favourable prognosis in patients with AC [hazard ratio, 0.56 (95% CI 0.38–0.83); p = 0.008]. We conclude that TTF1 amplification is a mechanism of high‐level TTF1 expression in a subset of NSCLCs. When expressed at high levels, this routinely used diagnostic marker is also an independent biomarker of favourable prognosis in AC. Copyright

Periostin is up-regulated in high grade and high stage prostate cancer

BackgroundExpression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far.MethodsHere, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed.ResultsIn total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05).ConclusionsOur data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.

L1CAM protein expression is associated with poor prognosis in non-small cell lung cancer

BackgroundThe L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition.ResultsL1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown.ConclusionsA subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion.

Frequent PD-L1 expression in testicular germ cell tumors.

Background:Many testicular germ cell cancers are curable despite metastatic disease, but about 10–15% of patients fail cisplatin-based first-line treatment. Immunotherapy is considered as additional treatment approach for these patients. Inhibition of the interaction between Programmed Death Receptor 1 (PD-1) and Programmed Death Receptor Ligand 1 (PD-L1) enhances T-cell responses in vitro and mediates clinical antitumour activity. We analysed the expression of PD-L1 in testicular germ cell tumours to evaluate its potential as target for immunotherapeutic strategies.Methods:Immunohistochemistry was performed in 479 formalin-fixed paraffin-embedded specimens using a rabbit monoclonal antibody (E1L3N). The tissue microarray consisted of 208 pure seminomas, 121 non-seminomas, 20 intratubular germ cell neoplasia unclassified (IGCNU) and 20 specimens of non-neoplastic testicular tissue.Results:Programmed Death Receptor Ligand-1 expression was found in 73% of all seminomas and in 64% of all non-seminomas. None of 20 IGCNU and none of 20 normal tissue specimens exhibited PD-L1 expression. PD-L1 positive stromal cells were only detected in seminomas, but not in non-seminomas. The anti PD-L1 antibody showed a pre-dominantly membranous staining pattern in testicular tumour cells, as well as expression in stromal cells.Conclusions:This frequent expression of PD-L1 in human testicular germ cell tumours suggests that patients with testicular germ cell tumours could profit from immunotherapeutic strategies using anti-PD1 and anti-PDL1 antibodies.

Primate genome architecture influences structural variation mechanisms and functional consequences

Significance Genomic structural variants (SVs) significantly contribute to human genetic variation and have been linked with numerous diseases. Compared with humans, the characterization of SVs occurring within and across nonhuman primates has lagged. We generated comprehensive massively parallel DNA sequencing-based SV maps in three nonhuman primate species and show that the rates of different SV formation mechanisms, such as nonallelic homologous recombination and Alu retrotransposition, vary significantly between the great apes and the rhesus macaque—leading to markedly different SV landscapes in these species. Linking gene expression data with species-specific gene duplications, we describe several instances where gene duplicates seem to lead to evolutionary innovation through the gain of gene expression in new tissues. Although nucleotide resolution maps of genomic structural variants (SVs) have provided insights into the origin and impact of phenotypic diversity in humans, comparable maps in nonhuman primates have thus far been lacking. Using massively parallel DNA sequencing, we constructed fine-resolution genomic structural variation maps in five chimpanzees, five orang-utans, and five rhesus macaques. The SV maps, which are comprised of thousands of deletions, duplications, and mobile element insertions, revealed a high activity of retrotransposition in macaques compared with great apes. By comparison, nonallelic homologous recombination is specifically active in the great apes, which is correlated with architectural differences between the genomes of great apes and macaque. Transcriptome analyses across nonhuman primates and humans revealed effects of species-specific whole-gene duplication on gene expression. We identified 13 gene duplications coinciding with the species-specific gain of tissue-specific gene expression in keeping with a role of gene duplication in the promotion of diversification and the acquisition of unique functions. Differences in the present day activity of SV formation mechanisms that our study revealed may contribute to ongoing diversification and adaptation of great ape and Old World monkey lineages.

Multicenter Immunohistochemical ALK-Testing of Non–Small-Cell Lung Cancer Shows High Concordance after Harmonization of Techniques and Interpretation Criteria

Introduction: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non–small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories. Methods: After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6×) and negativity (7×), as well as ALK positive-“borderline” character (2×), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed. Results: All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-“borderline” samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting. Conclusions: This so-called “ALK-Harmonization-Study” shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.

LACTB is a tumour suppressor that modulates lipid metabolism and cell state

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.