S1_Molecular&IHC method
Molecular and immunohistochemistry method
DNA extraction and quantification
Formalin-fixed and paraffine-embedded (FFPE) tumor samples were sliced in 5 µm-thick sections and manually micro-dissected under light microscope control to isolate the highest amount of tumor cellularity compared to contaminating non-neoplastic cells (hopefully 70% or more). Samples were treated with xylene and 100% ethanol to remove all wax traces and DNA was then isolated using the GeneRead DNA FFPE kit (Qiagen, Hilden, Germany, http://www.qiagen.com, catalogue n. 180134) according to manufacturer’s instructions. Subsequently, DNA amount and quality were controlled by means of NanoDrop platform (Invitrogen, Life Technologies, Foster City, CA, USA) following manufacturer’s details.
Next generation sequencing
For deep sequencing of multiple PCR amplicons of the genes under evaluation, the 50-gene Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies) with the Ion-Torrent™ Personal Genome Machine platform (Life Technologies) was used in all experiments. This panel is designed to amplify 207 amplicons covering about 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes recurrently mutated in human cancers (ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL), with wide coverage of KRAS, BRAF and EGFR genes frequently altered in lung cancer. Details of the target regions for the gene panel to assess in this LCC series are available at the website http://tools.lifetechnologies.com/downloads/cms_106003.csv.
A detailed description of all NGS technical procedures is reported in Supplemental File X. Genomic DNA samples (10 ng/ml) were used for all amplification reactions in the Ion AmpliSeq Library Kit2.0 (Life Technologies) according to the manufacturer’s instructions (MAN0006735 rev 5.0). Amplicons were ligated to P1 and barcode adapters using DNA ligase. Barcoded libraries were purified using AMPure Beads XP (Beckman coulters) and PCR-amplified for a total of five cycles. After a second round of purification with AMPure Beads, the amplified libraries were sized and quality assessed using the Agilent Bio Analyzer DNA High Sensitivity kit (Agilent Technologies) and quantified using the Qbit dsDNA HS kit (Invitrogen, Life Technologies).
Emulsion PCR and sample enrichment were performed using the IonOne Touch 2 instrument according to the manufacturer’s instruction (Life Technologies). In particular, an input concentration of DNA library obtained with the first amplification step was added to the emulsion PCR master-mix and the Ion sphere particles (ISPs) and a double phase (oil/water) PCR was performed. ISPs were then recovered and template positive ISPs were enriched using Dynabeads MyOne Streptavidin C1 beads (Life Technologies). Chips model 316 were used to sequence samples on Ion Torrent PGM using the Ion-PGM 200 sequencing kit following the manufacturer’s instructions.
Data from the PGM sequencing were initially processed using the Ion Torrent platform-specific software Torrent Suite to generate sequence reads, alignment of the reads on the reference genome Hg19, trim adapter sequences, filter and remove poor signal-profile reads. The variant calling from the sequencing data was generated using the Variant Caller plug-in. Filtered variants were visually examined using the Integrative Genomic Viewer tool to taste their level of quality and confirm the variant presence on both “+” and “-“ strand. Finally resulting variations were annotated using the Ensemble Variant Effect Predictor pipeline 1, COSMIC database, dbSNP database and MyCancerGenome database (http://www.mycancergenome.org/). Information about on the distribution of mutated genes in lung cancer was obtained from on-line catalogues of somatic mutations, such as Cosmic (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), ClinVar of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/clinvar/), NCBI’s database Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=gene), and GeneCards of the Weizmann Institute of Science, Israel (http://www.genecards.org/).
DNA Sanger sequencing
To ensure diagnostic accuracy and provide an external quality control on T-NGS outputs, several genes routinely worked up in our laboratory for the needs of precision medicine and found to be mutated in the current investigation, including EGFR (exons 18 to 21), TP53 (exons 5, 6, 7, 8, 10), BRAF (exon 11 and 15), KRAS (exons 2 to 4), NRAS (exons 2 to 4), PIK3CA (exons 1, 4, 7, 9, 10, 20, 21), RET (exons 8, 10, 11, 13-16) and STK11 (exons 1 to 10), were subjected to Sanger direct re-sequencing analysis by means of specific primer-driven and PCR-amplified sequences on 3500-Dx UE-IVD Genetic Analyzer (Life Technologies) according to previously refined molecular procedures 2.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) analysis was performed counting at least 100 tumor cells in 3m-thick paraffin sections. For FGFR1 gene, a commercially available break-apart, dual-color gene specific probe at 8p11 (Kreatech™ FISH Probes, Kreatech Diagnostics, Amsterdam, The Netherlands) was used according to manufacturer’s instructions (with minor modifications). FGFR1 amplification was defined by one of the following conditions as described by Schildhaus et al 3: the average number of FGFR1 signals per tumor cell nucleus was ≥ 6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters was ≥10%, or the percentage of tumor cells containing ≥5 FGFR1 signals was ≥50%.
Briefly, 3μm thick sections were cut from paraffin blocks and mounted on positively charged slides. Slides were heated overnight (56°C) and the day after de-waxed in xylene, treated with an ethanol to water series (100%-85%-70%), incubated in TE solution (TRIS 5mM-EDTA 1mM) at 96°C for 15 min, rinsed in distilled water, digested with pepsin (0,4%) in 0.01N HCl at 37°C from 6 to 10 min, washed again in distilled water, dehydrated in ethanol (96%), air dried, covered with coverslips, sealed, co-denatured in Hybridizer (Dako) at 80°C for 5 min and then incubated overnight at 37°C. The following day, coverslips were removed and the slides were washed for 2 min at 73°±1C in 2XSSC/0.3%NP40 and up to 1 min at room temperature in 2XSSC/0.1%NP40. The slides were allowed to dry in the dark at room temperature and nuclei were counterstained in Vectashield Antifade solution with DAPI (Vector laboratories, Inc. Burlingame CA). Tumor sections were then examined with LeicaDM600B florescence microscope (Leica, Wetzlar, Germany) equipped with a 100W mercury lamp and band pass filters specific for spectrum Orange, Spectrum Green and DAPI. Representative FISH images were captured with the automated cytogenetics platform CytoVision® (Leica, Germany).
Immunohistochemistry
The list of the antibodies used in the current study and the basic technical specifications are summarized in Table 2. All LCC samples were assessed for p40, a highly specific biomarker of SQC, which is consistently absent in lung ADC 4-7, and for TTF1, a determinant of lung and thyroid differentiation, which reacts with most but not all lung ADC 8, 9. Two different antibodies to p40, either monoclonal or polyclonal, were applied to the study. Tumors were also tested for cytokeratin 7 (CK7) and p63, two other widely agreed-upon biomarkers of ADC and SQC, respectively 10-15, and for p53 to dynamically compare protein expression with gene status. Briefly, 3- to 4-mm thick sections were made to react with the relevant antibodies and then incubated with a commercially available detection kit (EnVision™ FLEX+; Dako, Glostrup, Denmark) following the manufacturer’s instructions according to previously refined IHC procedures 6, 12. Results were rendered semiquantitatively as the percentage of immunoreactive cells, taking into account the entire tumor area on paraffin blocks and the relevant cellular compartmentalization (nuclear area for TTF1, p40, p53 and p63; cytoplasmic domain for synaptophysin, CK7 and vimentin). For defining diagnostic categories, TTF1 IHC was simply dichotomized into negative and positive tumors (the latter, whatever its extent), whereas p40 expression was divided into negative, positive and focally positive tumors, the latter figure resulting from scattered (<10%) tumor cells showing nuclear decoration (henceforth, simply heralded by the symbol “±”). In particular, TTF1-/p40- tumor category was defined as null phenotype because of the lack of diagnostic biomarkers for ADC or SQC, whereas TTF1-/p40± category was defined as unclear phenotype because of the focal occurrence of a squamous differentiation biomarker in otherwise TTF1 negative tumors.
References
(for S1_Molecular&IHC method only)
1. McLaren W, Pritchard B, Rios D, et al. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics 2010;26:2069-70.
2. Pelosi G, Gasparini P, Cavazza A, et al. Multiparametric molecular characterization of pulmonary sarcomatoid carcinoma reveals a nonrandom amplification of anaplastic lymphoma kinase (ALK) gene. Lung Cancer 2012;77:507-14.
3. Schildhaus HU, Heukamp LC, Merkelbach-Bruse S, et al. Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer. Mod Pathol 2012;25:1473-80.
4. Bishop JA, Teruya-Feldstein J, Westra WH, et al. p40 (DeltaNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol 2012;25:405-15.
5. Nonaka D. A Study of DeltaNp63 Expression in Lung Non-Small Cell Carcinomas. Am J Surg Pathol 2012
6. Pelosi G, Fabbri A, Bianchi F, et al. DeltaNp63 (p40) and thyroid transcription factor-1 immunoreactivity on small biopsies or cellblocks for typing non-small cell lung cancer: a novel two-hit, sparing-material approach. J Thorac Oncol 2012;7:281-90.
7. Pelosi G, Rossi G, Cavazza A, et al. DeltaNp63 (p40) distribution inside lung cancer: a driver biomarker approach to tumor characterization. Int J Surg Pathol 2013;21:229-39.
8. Pelosi G, Fraggetta F, Pasini F, et al. Immunoreactivity for thyroid transcription factor-1 in stage I non-small cell lung carcinomas of the lung. Am J Surg Pathol 2001;25:363-72.
9. Lazzaro D, Price M, De Felice M, Di Lauro R. The transcription factor, TTF-1, is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of fetal brain. Development 1991;113:1093-104.
10. Mukhopadhyay S, Katzenstein AL. Subclassification of Non-small Cell Lung Carcinomas Lacking Morphologic Differentiation on Biopsy Specimens: Utility of an Immunohistochemical Panel Containing TTF-1, Napsin A, p63, and CK5/6. Am J Surg Pathol 2011;35:15-25.
11. Pelosi G, Pasini F, Olsen Stenholm C, et al. p63 immunoreactivity in lung cancer: yet another player in the development of squamous cell carcinomas? J Pathol 2002;198:100-9.
12. Pelosi G, Rossi G, Bianchi F, et al. Immunhistochemistry by Means of Widely Agreed-Upon Markers (Cytokeratins 5/6 and 7, p63, Thyroid Transcription Factor-1, and Vimentin) on Small Biopsies of Non-small Cell Lung Cancer Effectively Parallels the Corresponding Profiling and Eventual Diagnoses on Surgical Specimens. J Thorac Oncol 2011;6:1039-49.
13. Rossi G, Papotti M, Barbareschi M, Graziano P, Pelosi G. Morphology and a limited number of immunohistochemical markers may efficiently subtype non-small-cell lung cancer. J Clin Oncol 2009;27:e141-2; author reply e3-4.
14. Terry J, Leung S, Laskin J, et al. Optimal immunohistochemical markers for distinguishing lung adenocarcinomas from squamous cell carcinomas in small tumor samples. Am J Surg Pathol 2010;34:1805-11.
15. Wang BY, Gil J, Kaufman D, et al. P63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors. Hum Pathol 2002;33:921-6.