大野論文正誤表

Figure S1. DNA quality control. TapeStation profiles of gDNA isolated from FF and matching FFPE block tumor tissues from 5 lung ADC patients. In each profile, the DIN, indicative of gDNA degradation status, is also displayed (numerical assessment ranges from 10 for undamaged gDNA, to 1 for highly fragmented gDNA) (a). The Table reports the gDNA concentration (ng/ul) assessed by NanoDrop, Qubit, and TapeStation, and purity (260/280 and 260/230) (b). Additionally, AYR and DIN parameters, indicative of FFPE gDNA fragmentation status, evaluated by a multiple PCR assay and TapeStation respectively, are reported. Image of agarose gel 1 % shows the gDNA smears indicative of the different degradation status of FF and FFPE gDNAs (c). Figure S2. The workflow illustrates samples processing and WES data analysis for both exome enrichment platforms. (PDF 187 kb

Additional file 4: Table S9. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Coverage distribution across all the coding exons of the 623 cancer related genes in each library. For each gene, the table reports the number of coding RefSeq exons downloaded from UCSC, their presence within 21 commercial re-sequencing cancer panels and further four cancer genes databases. The coverage distribution across all coding exons was performed using the GATK DiagnoseTarget tool. For each WES capture platform we reported: the number of ‘critical’ exons (average depth of coverage < 10× for at least 20 % of the length of the interval and with insufficient median depth across all FF and FFPE libraries), the number of exon regions missed by the kit target design file, and the % of passed exons (average depth of coverage ≥ 10× for at least 20 % of the length of the interval). (XLSX 120 kb

Additional file 3: Table S7 and Table S8. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Table S7. Mean coverage achieved by Agilent SureSelect and Roche NimbleGen libraries within 90 PCR-capture amplicons. Mean coverage ± SD within 90 regions amplified by AmpliSeq Colon and Lung Cancer Panel v.1 (Thermo Fisher Scientific) from ‘FF’, ‘FFPE’ and ‘FF plus FFPE’ samples achieved by Agilent SureSelect and Roche NimbleGen libraries respectively. In each column, the mean coverage values are reported for each amplicon, and the heat map was created using two-color scale (lowest value is represented by dark blue and highest value by dark red). Table S8. Variant calling comparison between the two WES systems (Agilent SureSelect and Roche NimbleGen) and the AmpliSeq Colon and Lung Cancer Panel. List of FFPE and matched FF samples genetic variants called by VC v.4.2 plugin on Ion PGMTM data and GATK pipeline in both exome capture systems. All variants are annotated with gene ID, locus, reference sequence, variant allele according to the hg19 Reference Genome. The red bars show the variant allele frequency (%) detected by VC on Ion pipeline and GATK on both Agilent SureSelect and Roche NimbleGen WES (0* means variant not called but found by IGV visual inspection of BAM files). All variants are annotated for COSMIC or dbSNP (rs number) together with the codons involved and the amino acid change (AA). The 'Effect' column reports if the variant is in a coding region, discerning between nonsynonymous, synonymous and non-sense, or in an intron, downstream the gene or in a splicing region. The last four columns of the table reports the Minor Allele Frequency (MAF) reported in the 1000 Genomes Project, the prediction effect on the protein based on SIFT and Polyphen algorithms and the conservation score namely GERP. For SIFT prediction, the higher the number, the lower is the effect, whereas for Polyphen prediction is the opposite. Thus, a higher score for GERP indicates a higher conservation of the gene across 34 mammalian species. Abbreviation: - not available data. (XLSX 44 kb

Additional file 2: Table S1, Table S2, Table S3, Table S4, Table S5, and Table S6. of Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples

Table S1. Sequencing metrics for libraries prepared with both Agilent SureSelect XT v.5 and Roche NimbleGen v.3.0  kits starting from five matched FF and FFPE tumor samples. Table S2. Variant detection comparison between matched FF-FFPE pairs. For each matched FF-FFPE pair, the number and the percentage of both SNVs and InDels common to both sample types, and unique to either FF or FFPE sample are reported. Table S3. Genotype CR and NRDR between matched FF-FFPE pairs at increasing coverage thresholds. For each matched FF-FFPE pair, the genotype CR was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples (a). For each matched FF-FFPE pair, the NRDR was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples (b). Table S4. Genotype CR and NRDR between matched FF-FFPE pairs computed for each transition type at increasing coverage thresholds. For each matched FF-FFPE pair, the genotype CR for each transition type was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples; p-values for two-tail t-test for each comparison between two transition types are reported at the bottom of the table (a). For each matched FF-FFPE pair, the NRDR for each transition type was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both samples; p-values for two-tail t-test for each comparison between two transition types are reported at the bottom of the table (b). Table S5. Variant detection comparison between exome libraries prepared with both Agilent SureSelect and Roche NimbleGen kit. The table reports the total number and the percentage of SNVs and InDels common to both library prep types for each sample, and unique to either Agilent SureSelect and Roche NimbleGen kit. The comparison was performed considering both the whole kit-specific target region and the 42 Mb of common target region. Table S6. Genotype CR and NRDR rates within the shared 42 Mb target region between Agilent SureSelect and Roche NimbleGen at increasing coverage thresholds. For each sample, the genotype CR was computed as the ratio between the sum of concordant genotypes and the sum of all genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both Agilent SureSelect and Roche NimbleGen libraries (a). For each sample, the NRDR was computed as the ratio between the sum of non-concordant genotypes and the sum of all non-reference genotypes called at genomic positions covered at least a certain coverage threshold (from 1 to 50×) in both in both Agilent SureSelect and Roche NimbleGen libraries (b). (XLSX 54 kb

Image_2_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

DataSheet_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.docx

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

Image_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p