Jörg Sänger

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell Lung Cancer

FGFR1 amplification provides a therapeutic target for squamous cell lung cancer, which is resistant to other targeted lung cancer drugs. A Smoking Gun for Lung Cancer Detectives and scientists alike need strong evidence to take their cases to the judge, who for scientists is often a patient with a deadly disease. Yet, new culprits are sometimes found that can break a case wide open. Lung cancer, which accounts for more than 10% of the global cancer burden, has a poor prognosis and inadequately responds to chemotherapy and radiotherapy. New targeted treatments for lung adenocarcinomas inhibit the oncogenic versions of signaling protein kinases that arise from mutations typically found in lung cancer patients who have never smoked. However, smokers frequently suffer from a different deviant, squamous cell lung cancers, for which there are no known molecular genetic targets for therapy. Now, Weiss et al. have fingered a new suspect in smoking-related lung cancer: amplification of the FGFR1 gene, which encodes the fibroblast growth factor receptor 1 tyrosine kinase (FGFR1). To identify therapeutically viable genetic alterations that may influence squamous cell lung cancer, Weiss et al. performed genomic profiles on a large set of lung cancer specimens. Squamous cell lung cancer samples showed FGFR1 amplification, which was not found in other lung cancer subtypes. The authors then determined that a molecule that broadly inhibits FGF receptor function could block tumor growth and cause cell death in the cancers that expressed high amounts of the FGFR1 gene product in a manner that was dependent on FGFR1 expression. Moreover, FGFR1 inhibition resulted in a considerable decrease in tumor size in a mouse model of FGFR1-amplified lung cancer. This culmination of evidence implies that inhibition of this receptor tyrosine kinase should be explored as a candidate therapy for corralling squamous cell lung cancer in smokers. Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

CD74-NRG1 fusions in lung adenocarcinoma

UNLABELLED We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP‐6 is involved in numerous developmental processes. We have demonstrated expression of BMP‐6 in breast cancer cell lines by RT‐PCR and immuno‐histochemistry. The level of BMP‐6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP‐6 mRNA levels in a dose‐dependent manner, with a maximum at 50 ng/ml EGF under serum‐free conditions in hormone‐sensitive (MCF‐7) and in hormone‐insensitive (SK‐BR‐3) breast cancer cell lines. The EGF‐like growth factors transforming growth factor‐α, amphiregulin and betacellulin were also able to elevate the BMP‐6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG1517 repressed the inductive effect of these growth factors, indicating an EGF receptor‐mediated regulation of BMP‐6 mRNA. In addition, BMP‐6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor‐free resection margins. In 12 of these 18 patients, at least a 10‐fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor‐free samples was observed. This suggests a putative relationship between EGF receptor and BMP‐6 mRNA levels in breast cancer. Int. J. Cancer 80:250–256, 1999.

Elevated activity and expression of Src-family kinases in human breast carcinoma tissue versus matched non-tumor tissue

Abstract Src-family kinase expression was measured in 52 human mammary tumor (T) specimens compared with non-tumor (NT) tissue from the same patient by enzymatic assays employing a Src-kinase family-specific peptide substrate and by immunoblotting with an antibody recognizing the Src-family kinases Src, Fyn, and Yes. In the T specimens, the mean enzymatic activity was moderately elevated (T: 160 fmol ATP min−1 mg−1; NT: 115 fmol ATP min−1 mg−1) with 25 tumor samples having higher activity than the corresponding NT tissue, 17 having lower activity, and no activity detectable in ten T/NT pairs. Immunoblotting revealed clearly elevated expression in 25 tumor tissues and no differences or expression below the detection limit in the remaining T/NT pairs. The data are in agreement with a possible role of Src-family kinases for the biology of mammary carcinoma.

Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.

Inverse expression of somatostatin and CXCR4 chemokine receptors in gastroenteropancreatic neuroendocrine neoplasms of different malignancy.

Introduction Somatostatin receptors (SSTR) are widely distributed in well-differentiated neuroendocrine neoplasms (NEN) and serve as primary targets for diagnostics and treatment. An overexpression of the chemokine receptor CXCR4, in contrast, is considered to be present mainly in highly proliferative and advanced tumors. Comparative data are still lacking, however, for neuroendocrine carcinomas (NEC). Methods SSTR subtype (1, 2A, 3, 5) and CXCR4 expression was evaluated in G1 (n = 31), G2 (n = 47), and low (G3a; Ki-67: 21–49%; n = 21) and highly proliferative (G3b; Ki-67: >50%, n = 22) G3 (total n = 43) gastroenteropancreatic NEN samples by performing immunohistochemistry with monoclonal rabbit anti-human anti-SSTR and anti-CXCR4 antibodies, respectively, and was correlated with clinical data. Results Both CXCR4 and SSTR were widely expressed in all tumors investigated. CXCR4 expression differed significantly between the G1 and G3 specimens and within the G3 group (G3a to G3b), and was positively correlated with Ki-67 expression. SSTR2A, in contrast, exhibited an inverse association with Ki-67. SSTR2A was highly expressed in G1 and G2 tumors, but was significantly less abundant in G3 carcinomas. Additionally, SSTR1 expression was higher in G3a than in G3b tumors. Conclusion We observed an elevation in CXCR4 and a decrease in SSTR2A expression with increasing malignancy. Interestingly, 23% of the G3 specimens had strong SSTR2A expression. Because CXCR4 was strongly expressed in highly proliferative G3 carcinomas, it is an interesting new target and needs to be validated in larger studies.

Molecular imaging of late somatostatin receptor-positive metastases of renal cell carcinoma in the pancreas by 68Ga DOTATOC PET/CT: a rare differential diagnosis to multiple primary pancreatic neuroendocrine tumors.

Ga somatostatin receptor PET/CT, currently the most sensitive imaging modality for well-differentiated neuroendocrine tumors, is based on the molecular imaging of somatostatin receptors (SSTRs) that are expressed in different tumor entities such as neuroendocrine neoplasms, lymphomas, meningiomas, or renal cell cancer (RCC). Most neuroendocrine neoplasms show a high expression of SSTR subtypes 2A and 5, whereas the overexpression of SSTR2A in RCC is mainly seen in peritumoral vessels. Here we report a case with strongly SSTR-positive pancreatic lesions detected by Ga DOTATOC PET/CT, which histologically turned out to be ultralate metastases of a RCC.

Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In this regard, we compared the expression of three proliferation markers, Ki-67, Topoisomerase II alpha (TOP2A), and RacGAP1, in a series of tumor samples from 104 BP-NEN patients (24 TC, 21 AC, 52 SCLC, 7 LCNEC) using different evaluation methods (immunohistochemistry (IHC): Average evaluation, Hotspot evaluation, digital image analysis; RT-qPCR). The results indicated that all three markers had increased protein and mRNA expression with poorer differentiation and correlated well with each other, as well as with grading, staging, and poor survival. Compared with Ki-67 and TOP2A, RacGAP1 allowed for a clearer prognostic statement. The cut-off limits obtained for Ki-67-Average (IHC) were TC-AC 1.5, AC-SCLC 19, and AC-LCNEC 23.5. The Hotspot evaluation generated equal to higher, the digital image analysis generally lower between-entity cut-off limits. All three markers enabled a clear-cut differentiation between the BP-NEN entities, and all methods evaluated were suitable for marker assessment. However, to define optimal cut-off limits, the Ki-67 evaluation methods should be standardized. RacGAP1 appeared to be a new marker with great potential.

Localization of Radiolabeled Somatostatin Analogs in the Spleen.

Radiolabeled somatostatin (SST) analogs, used to visualize and treat SST receptor (SSTR)-expressing neuroendocrine tumors, also accumulate in the spleen. There is a high interpatient variation and no significant radiation-induced splenic toxicity; however, an absorbed dose-related reduction in spleen size was detected. However, the exact localization of radioactivity and the role of SST receptors in splenic retention are unknown. Therefore, we performed ex vivo micro-SPECT of the isolated spleen from a patient with a pancreatic neoplasm after 1 GBq (27 mCi) Lu-DOTATATE administration, followed by autoradiography and immunohistochemistry. Ex vivo autoradiography demonstrated convincingly that most radioactivity accumulated in red pulp.