Comparative proteomics reveals a diagnostic signature for pulmonary head‐and‐neck cancer metastasis
2018; Springer Nature; Volume: 10; Issue: 9 Linguagem: Inglês
10.15252/emmm.201708428
ISSN1757-4684
AutoresHanibal Bohnenberger, Lars Kaderali, Philipp Ströbel, Diego Yepes, Uwe Plessmann, Neekesh V. Dharia, Sha Yao, Carina Heydt, Sabine Merkelbach‐Bruse, Alexander Emmert, Jonatan Hoffmann, Julius Bodemeyer, Kirsten Reuter‐Jessen, Anna‐Maria Lois, Leif H. Dröge, Philipp Baumeister, Christoph Walz, Lorenz Biggemann, Roland B. Walter, Björn Häupl, Federico Comoglio, Kuan‐Ting Pan, Sebastian Scheich, Christof Lenz, Stefan Küffer, Felix Bremmer, Julia Kitz, Maren Sitte, Tim Beißbarth, Marc Hinterthaner, Martin Sebastian, Joachim Lotz, Hans‐Ulrich Schildhaus, Hendrik A. Wolff, Bernhard C. Danner, Christian Brandts, Reinhard Büttner, Martin Canis, Kimberly Stegmaier, Hubert Serve, Henning Urlaub, Thomas Oellerich,
Tópico(s)RNA modifications and cancer
ResumoResearch Article10 August 2018Open Access Transparent process Comparative proteomics reveals a diagnostic signature for pulmonary head-and-neck cancer metastasis Hanibal Bohnenberger Hanibal Bohnenberger Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Lars Kaderali Lars Kaderali Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany Search for more papers by this author Philipp Ströbel Philipp Ströbel Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Diego Yepes Diego Yepes Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Uwe Plessmann Uwe Plessmann Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Neekesh V Dharia Neekesh V Dharia orcid.org/0000-0001-5048-067X Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Division of Pediatric Hematology/Oncology, Boston Childrens Hospital, Boston, MA, USA The Broad Institute, Cambridge, MA, USA Search for more papers by this author Sha Yao Sha Yao Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Carina Heydt Carina Heydt Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Sabine Merkelbach-Bruse Sabine Merkelbach-Bruse Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Alexander Emmert Alexander Emmert Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Jonatan Hoffmann Jonatan Hoffmann Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Julius Bodemeyer Julius Bodemeyer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Kirsten Reuter-Jessen Kirsten Reuter-Jessen Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Anna-Maria Lois Anna-Maria Lois Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Leif Hendrik Dröge Leif Hendrik Dröge Department of Radiooncology, University Medical Center, Göttingen, Germany Search for more papers by this author Philipp Baumeister Philipp Baumeister Department of Otorhinolaryngology, University Hospital Munich, Ludwig-Maximilian-University München, München, Germany Search for more papers by this author Christoph Walz Christoph Walz Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany Search for more papers by this author Lorenz Biggemann Lorenz Biggemann Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany Search for more papers by this author Roland Walter Roland Walter Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Björn Häupl Björn Häupl Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Federico Comoglio Federico Comoglio Department of Haematology, University of Cambridge, Cambridge, UK Cambridge Institute for Medical Research, Wellcome Trust/MRC Stem Cell Institute, Cambridge, UK Search for more papers by this author Kuan-Ting Pan Kuan-Ting Pan Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sebastian Scheich Sebastian Scheich Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Christof Lenz Christof Lenz Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Bioanalytics, University Medical Center, Göttingen, Germany Search for more papers by this author Stefan Küffer Stefan Küffer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Felix Bremmer Felix Bremmer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Julia Kitz Julia Kitz Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Maren Sitte Maren Sitte Institute of Medical Statistics, University Medical Center, Göttingen, Germany Search for more papers by this author Tim Beißbarth Tim Beißbarth Institute of Medical Statistics, University Medical Center, Göttingen, Germany Search for more papers by this author Marc Hinterthaner Marc Hinterthaner Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Martin Sebastian Martin Sebastian Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Joachim Lotz Joachim Lotz Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany German Cardiovascular Research Center, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK), Partnersite Göttingen, Germany Search for more papers by this author Hans-Ulrich Schildhaus Hans-Ulrich Schildhaus Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Hendrik Wolff Hendrik Wolff University Medical Center, Göttingen, Germany Department of Radiooncology, University Medical Center, Regensburg, Germany Search for more papers by this author Bernhard C Danner Bernhard C Danner Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Christian Brandts Christian Brandts Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Reinhard Büttner Reinhard Büttner Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Martin Canis Martin Canis Department of Otorhinolaryngology, University Hospital Munich, Ludwig-Maximilian-University München, München, Germany Search for more papers by this author Kimberly Stegmaier Kimberly Stegmaier Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Division of Pediatric Hematology/Oncology, Boston Childrens Hospital, Boston, MA, USA The Broad Institute, Cambridge, MA, USA Search for more papers by this author Hubert Serve Hubert Serve Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Henning Urlaub Henning Urlaub Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany Search for more papers by this author Thomas Oellerich Corresponding Author Thomas Oellerich [email protected] orcid.org/0000-0001-6570-6125 Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Hanibal Bohnenberger Hanibal Bohnenberger Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Lars Kaderali Lars Kaderali Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany Search for more papers by this author Philipp Ströbel Philipp Ströbel Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Diego Yepes Diego Yepes Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Uwe Plessmann Uwe Plessmann Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Neekesh V Dharia Neekesh V Dharia orcid.org/0000-0001-5048-067X Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Division of Pediatric Hematology/Oncology, Boston Childrens Hospital, Boston, MA, USA The Broad Institute, Cambridge, MA, USA Search for more papers by this author Sha Yao Sha Yao Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Carina Heydt Carina Heydt Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Sabine Merkelbach-Bruse Sabine Merkelbach-Bruse Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Alexander Emmert Alexander Emmert Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Jonatan Hoffmann Jonatan Hoffmann Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Julius Bodemeyer Julius Bodemeyer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Kirsten Reuter-Jessen Kirsten Reuter-Jessen Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Anna-Maria Lois Anna-Maria Lois Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Leif Hendrik Dröge Leif Hendrik Dröge Department of Radiooncology, University Medical Center, Göttingen, Germany Search for more papers by this author Philipp Baumeister Philipp Baumeister Department of Otorhinolaryngology, University Hospital Munich, Ludwig-Maximilian-University München, München, Germany Search for more papers by this author Christoph Walz Christoph Walz Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany Search for more papers by this author Lorenz Biggemann Lorenz Biggemann Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany Search for more papers by this author Roland Walter Roland Walter Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Björn Häupl Björn Häupl Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Federico Comoglio Federico Comoglio Department of Haematology, University of Cambridge, Cambridge, UK Cambridge Institute for Medical Research, Wellcome Trust/MRC Stem Cell Institute, Cambridge, UK Search for more papers by this author Kuan-Ting Pan Kuan-Ting Pan Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Sebastian Scheich Sebastian Scheich Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Christof Lenz Christof Lenz Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Bioanalytics, University Medical Center, Göttingen, Germany Search for more papers by this author Stefan Küffer Stefan Küffer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Felix Bremmer Felix Bremmer Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Julia Kitz Julia Kitz Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Maren Sitte Maren Sitte Institute of Medical Statistics, University Medical Center, Göttingen, Germany Search for more papers by this author Tim Beißbarth Tim Beißbarth Institute of Medical Statistics, University Medical Center, Göttingen, Germany Search for more papers by this author Marc Hinterthaner Marc Hinterthaner Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Martin Sebastian Martin Sebastian Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany Search for more papers by this author Joachim Lotz Joachim Lotz Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany German Cardiovascular Research Center, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK), Partnersite Göttingen, Germany Search for more papers by this author Hans-Ulrich Schildhaus Hans-Ulrich Schildhaus Institute of Pathology, University Medical Center, Göttingen, Germany Search for more papers by this author Hendrik Wolff Hendrik Wolff University Medical Center, Göttingen, Germany Department of Radiooncology, University Medical Center, Regensburg, Germany Search for more papers by this author Bernhard C Danner Bernhard C Danner Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany Search for more papers by this author Christian Brandts Christian Brandts Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Reinhard Büttner Reinhard Büttner Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany Search for more papers by this author Martin Canis Martin Canis Department of Otorhinolaryngology, University Hospital Munich, Ludwig-Maximilian-University München, München, Germany Search for more papers by this author Kimberly Stegmaier Kimberly Stegmaier Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Division of Pediatric Hematology/Oncology, Boston Childrens Hospital, Boston, MA, USA The Broad Institute, Cambridge, MA, USA Search for more papers by this author Hubert Serve Hubert Serve Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Henning Urlaub Henning Urlaub Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany Search for more papers by this author Thomas Oellerich Corresponding Author Thomas Oellerich [email protected] orcid.org/0000-0001-6570-6125 Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany Search for more papers by this author Author Information Hanibal Bohnenberger1,‡, Lars Kaderali2,‡, Philipp Ströbel1, Diego Yepes3,4, Uwe Plessmann5, Neekesh V Dharia6,7,8, Sha Yao1, Carina Heydt9, Sabine Merkelbach-Bruse9, Alexander Emmert10, Jonatan Hoffmann1, Julius Bodemeyer1, Kirsten Reuter-Jessen1, Anna-Maria Lois1, Leif Hendrik Dröge11, Philipp Baumeister12, Christoph Walz13, Lorenz Biggemann14, Roland Walter3, Björn Häupl3,4, Federico Comoglio15,16, Kuan-Ting Pan5, Sebastian Scheich3, Christof Lenz5,17, Stefan Küffer1, Felix Bremmer1, Julia Kitz1, Maren Sitte18, Tim Beißbarth18, Marc Hinterthaner10, Martin Sebastian3, Joachim Lotz14,19, Hans-Ulrich Schildhaus1, Hendrik Wolff20,21, Bernhard C Danner10, Christian Brandts3,4, Reinhard Büttner9, Martin Canis12, Kimberly Stegmaier6,7,8, Hubert Serve3,4, Henning Urlaub5,14 and Thomas Oellerich *,3,4 1Institute of Pathology, University Medical Center, Göttingen, Germany 2Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany 3Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany 4German Cancer Research Center, German Cancer Consortium, Heidelberg, Germany 5Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 6Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 7Division of Pediatric Hematology/Oncology, Boston Childrens Hospital, Boston, MA, USA 8The Broad Institute, Cambridge, MA, USA 9Institute of Pathology, University Hospital Cologne, University of Cologne, Köln, Germany 10Department of Thoracic and Cardiovascular Surgery, University Medical Center, Göttingen, Germany 11Department of Radiooncology, University Medical Center, Göttingen, Germany 12Department of Otorhinolaryngology, University Hospital Munich, Ludwig-Maximilian-University München, München, Germany 13Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany 14Institute for Diagnostic and Interventional Radiology, University Medical Center, Göttingen, Germany 15Department of Haematology, University of Cambridge, Cambridge, UK 16Cambridge Institute for Medical Research, Wellcome Trust/MRC Stem Cell Institute, Cambridge, UK 17Bioanalytics, University Medical Center, Göttingen, Germany 18Institute of Medical Statistics, University Medical Center, Göttingen, Germany 19German Cardiovascular Research Center, Deutsches Zentrum für Herz-Kreislaufforschung (DZHK), Partnersite Göttingen, Germany 20University Medical Center, Göttingen, Germany 21Department of Radiooncology, University Medical Center, Regensburg, Germany ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +496963016148; E-mail: [email protected] EMBO Mol Med (2018)10:e8428https://doi.org/10.15252/emmm.201708428 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC. Synopsis Differentiation between head-and-neck cancer metastasis and primary lung cancer is clinically important for therapy selection. A novel diagnostic proteomic signature allows differentiation between these tumour types, and a proteomic resource for squamous cell tumours is here provided. The protein expression profiles of 63 squamous cell lung and 49 head-and-neck tumours were analysed by quantitative mass spectrometry yielding a proteomic resource covering 6,214 quantified proteins. 518 proteins were found to be differentially expressed between squamous cell lung and head-and-neck cancers which are known to share genomic and morphological features. A diagnostic proteomic signature for differentiation between primary squamous cell lung cancers and head-and-neck cancer metastases was identified by quantitative mass-spectrometry-based proteomics and validated in independent patient cohorts. Introduction Head-and-neck squamous cell carcinoma (HNSCC) affects more than 500,000 patients every year worldwide (Ferlay et al, 2010; Jemal et al, 2011; Chaturvedi et al, 2013). Besides local recurrences, long-term survival of patients with resectable HNSCC is limited by frequently occurring distant metastases, of which up to 80% occur in the lung (Calhoun et al, 1994; Jones et al, 1995; de Bree et al, 2000; Ferlito et al, 2001; Liao et al, 2007). Thus, patients with HNSCC are screened for lung metastasis after tumour resection (de Bree et al, 2000; Merkx et al, 2002). However, because tobacco-smoking is not only a major risk factor for the development of HNSCC, but also for lung cancer, HNSCC patients are furthermore at high risk of developing metachronous squamous cell carcinomas of the lung (SQCLC; Talamini et al, 2002). Differentiation between lung metastasis of HNSCC (metHNSCC) and SQCLC is of clinical importance, as the diagnosis guides the therapeutic procedures that can range from curative treatment for SQCLC patients to palliative treatment for patients with metastatic HNSCC (Atabek et al, 1987; Jacobs et al, 1992; Jones et al, 1995; Kuriakose et al, 2002; Henschke et al, 2003; Battafarano et al, 2004; Wisnivesky et al, 2004; Pignon et al, 2008; Shiono et al, 2009). Despite this clinical importance, reliable biomarkers for differentiation between SQCLC and HNSCC metastasis in the lung are currently lacking; this may be due, at least partly, to the overlapping aetiology, morphology and biology of these tumour entities. Because decision-making by clinicians currently relies on non-validated clinical and imaging criteria, there is an urgent need for reliable molecular biomarkers that can differentiate between SQCLC and HNSCC lung metastases (Geurts et al, 2005). Head-and-neck squamous cell carcinoma and SQCLC have been extensively studied at the genomic and transcriptomic levels. However, in addition to their shared morphology, HNSCC and SQCLC exhibit largely overlapping patterns of genetic mutations and copy number alterations (van Oijen et al, 2000; Tabor et al, 2002; Geurts et al, 2005, 2009; Talbot et al, 2005; Vachani et al, 2007; Cancer Genome Atlas Research, 2012; Cancer Genome Atlas, 2015; Ichinose et al, 2016a). Given the apparent inability of genomic features to differentiate reliably between HNSCC and SQCLC, we have investigated a clinically and genetically well-characterised cohort of SQCLC, HNSCC and undetermined lung tumours by quantitative mass spectrometry-based proteomics. We reasoned that this proteomic approach, rather than genomic and transcriptomic studies, may identify a suitable biomarker panel and may also provide some more general insights into the biology of these tumours. Results Comparative proteomic characterisation of squamous cell carcinomas of the lung and the head-and-neck region Differentiation between metHNSCC and primary SQCLC is of fundamental clinical importance for therapeutic stratification. However, diagnostic biomarkers are so far lacking, owing to the large number of morphological and genomic features that are shared by these tumour entities. Because the two tumour types have not been systematically compared at the proteome level, we wished to explore the possibility of identifying proteomic diagnostic biomarkers. To this end, we characterised the protein expression profiles of 44 formalin-fixed and paraffin-embedded (FFPE) SQCLC tissues and 30 FFPE HNSCC tissues from patients who developed squamous cell tumours in the lung in the course of their disease. All SQCLCs in our cohort were in tumour stage I–III with grades 2–3, and the tumour stage of HNSCCs ranged from I to IV with grades 2–3 (see Table 1 and Dataset EV1 for detailed patient characteristics). All patients were treated by surgery and 40 of them had received additional adjuvant (chemo)radiotherapy. None of the patients had received neoadjuvant therapy or primary chemoradiotherapy. Squamous cell histology of all samples was confirmed by expert pathology review including immunohistochemical staining of the markers p63 and cytokeratin 5/6 (Fig 1A). Moreover, targeted next-generation sequencing revealed similar mutation patterns in both HNSCC and SQCLC samples from our cohort (Fig 1B). This is in accordance with genomic studies that have reported similar somatic mutation patterns and frequencies in both diseases, as illustrated in Fig 1C (Cancer Genome Atlas Research N, 2012; Cancer Genome Atlas, 2015). The mutations detected affected mostly the PI3-kinase (PI3K) and Ras pathways, receptor tyrosine kinases (RTK), TP53 and the NFE2L2/KEAP1 pathway (Fig 1B and Dataset EV1). Notably, three HNSCC samples were positive for human papillomavirus (HPV) 16. Table 1. Patient characteristics training cohort Characteristic Squamous cell lung carcinoma (n = 44) Squamous cell head-and-neck carcinoma (n = 30) Age median (range) [years] 66 (49–81) 55 (31–76) Male sex [no. (%)] 32 (72.7) 26 (86.7) Smoking or tobacco use [no. (%)] Current or former 22 (50.0) 28 (93.3) Never 0 (0.0) 2 (6.7) Not reported 22 (50.0) 0 (0.0) Site of primary tumour [no. (%)] Lung 44 (100.0) — Larynx — 12 (40.0) Oral cavity — 5 (16.7) Pharynx — 13 (43.4) Systemic therapy regimen [no. (%)] Adjuvant therapy 22 (50.0) 18 (60.0) Neoadjuvant therapy 0 (0.0) 0 (0.0) Overall survival: median follow-up time (range) [months] 19 (1–38) 57 (7–169) No. of reported deaths (%) 17 (31.8) 20 (66.7) Local relapse of HNSCC [no. (%)] — 5 (16.7) Time HNSCC until lung tumour: Median (range) [months] — 20 (1–121) Staging UICC (7th edition) Stage I [no. (%)] 14 (31.8) 1 (3.3) Stage II [no. (%)] 11 (25.0) 5 (16.7) Stage III [no. (%)] 19 (43.2) 7 (23.3) Stage IV [no. (%)] 0 (0.0) 17 (56.7) TP53 sequence Wild type [no. (%)] 12 (27.3) 12 (40.0) Mutated [no. (%)] 31 (70.5) 12 (40.0) Unknown 1 (2.2) 6 (20.0) Figure 1. Genetic comparison of lung and head-and-neck carcinomas A. Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous cell carcinomas. Scale bar: 500 μm. B. Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed. (Blue, no mutation/no HPV detected; red, mutation/HPV detected; white, no data available) C. Comparison of mutation rates in HNSCC (n = 279) and SQCLC (n = 178) cases from TCGA. Genes exhibiting a mutation rate > 0.25 in both cancer types are labelled. Download figure Download PowerPoint For the proteomic characterisation of the 74 tumour samples (44 SQCLC, 30 HNSCC), we combined a filter-aided sample preparation approach (FASP) with Super-SILAC-based quantitative mass spectrometry (Fig 2A; Wisniewski et al, 2009; Erde et al, 2014; Bohnenberger et al, 2015). Before protein extraction, tumour cell areas were marked under microscopic view by expert pathologists, and the tumour cell content was enriched to >80% by macrodissection, which is a well-established technique for the molecular analysis of tumour tissue samples in routine pathology diagnostics (Cree et al, 2014). After FASP-based protein extraction, the tumour-derived proteins were mixed in equimolar amounts with a Super-SILAC spike-in protein standard that was used for relative quantification of protein abundance. The Super-SILAC standard consisted of the four lung cancer cell lines NCI-H2228, HCC15, HCC44 and NCI-H1339. It represented adequately the proteomes of SQCLC and HNSCC samples, as reflected by the fact that more than 90% of the SILAC ratios were within a fivefold range, allowing accurate and comparable protein quantification (Fig 2B). The protein mixture was digested with the protease trypsin, and the resulting peptides were finally analysed by high-resolution mass spectrometry. Figure 2. Proteomic comparison of lung and head-and-neck carcinomas A. Schematic experimental workflow: Patient-derived tumour samples were lysed according to the FASP protocol; the resulting proteins were mixed in equimolar amounts with a Super-SILAC spike-in standard and digested with trypsin. The MS analysis of each sample was performed in duplicate, and data analysis was performed with the software packages MaxQuant, Perseus and R. B. Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples. C. Boxplot showing the numbers of quantified proteins, derived from 44 SQCLC and 30 HNSCC tissue samples. The central line in the boxes represents the median number of proteins over all samples, and upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum. D. Distribution of MS-based protein signal intensities in all 74 tissue samples of the training cohort. E. Principal component analysis of SQCLC (n = 44; blue) and HNSCC (n = 30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively. Download
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