Additional file 2: Figure S1. of PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples

H&E staining of representative sections for the six breast cancer samples analyzed by LMD-PAT-H-MS and classical PAT-H-MS. Scale bar = 10 mm. (PDF 1881 kb

Additional file 3: Dataset S2. of PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples

Dataset containing the measured AUCs and L/H ratios for the breast cancer samples shown in Figs. 3, 4, and 5 and Additional file 2: Figure S1. (XLSX 105 kb

DataSheet_1_Genomic and transcriptomic analyses of thyroid cancers identify DICER1 somatic mutations in adult follicular-patterned RAS-like tumors.pdf

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAFV600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients.MethodsHere, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA).ResultsThe occurrence of BRAFV600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1-mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database.ConclusionEven though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification.</p

Derivation and biochemical analysis of iPSC upon conditional <i>Ezh2</i> inactivation.

<p>A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×10³, 5×10³, 1×10⁴ or 6×10⁴ MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative <i>Ezh2</i> control (<i>+/+</i>) and mutant <i>(ΔSET/ΔSET</i>) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.</p

Effect of PRC2 inactivation on established <i>Ezh2^ΔSET/ΔSET</i> iPSC clones and TF–induced reprogramming.

<p>A. Western blot analysis of EED protein levels in two <i>Ezh2^ΔSET/ΔSET</i> iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting <i>Eed</i> (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two <i>Ezh2^ΔSET/ΔSET</i> iPSC clones. <i>Ezh2</i>-mutant iPSC were infected with viruses expressing two independent hairpins for <i>Eed</i> or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on <i>Cdkn2a^−/− Ezh2</i>-proficient TTF expressing three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×10³ (upper panel) <i>Cdkn2a^−/− Ezh2</i>-proficient TTF expressing three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP⁺ iPSC colonies. Column height represents number of AP⁺ iPSC colonies obtained from 1×10³ TTF expressing either one of the three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.</p

Characterization of pluripotency in iPSC clones reprogrammed upon <i>Ezh2</i> inactivation.

<p>A. Images of phase contrast (left), alkaline phosphatase staining (middle) and <i>Oct4</i>-driven GFP fluorescence (right) of representative control (<i>Ezh2^+/ΔSET</i>, upper row) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (<i>Ezh2^+/ΔSET</i>, upper row) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (<i>Ezh^+/+</i>, grey) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either <i>Ezh2</i> control (<i>Ezh2^+/+</i>) or mutant (<i>Ezh2^ΔSET/ΔSET</i>) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between <i>Ezh2^+/+</i> and <i>Ezh2^ΔSET/ΔSET</i> iPSC (left panel), <i>Ezh2^+/+</i> iPSCs and MEF (central panel) and <i>Ezh2^ΔSET/ΔSET</i> iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (<i>Ezh2^+/+</i>, first and second column) and two mutant (<i>Ezh2^ΔSET/ΔSET</i>, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.</p

Genome-wide distribution of H3K27me3 in <i>Ezh2^ΔSET/ΔSET</i> iPSC revealed through ChIP–seq.

<p>A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSCs. B. Venn diagram displaying overlap between H3K27me3⁺ genes in <i>Ezh2</i>^+/+ (grey) and <i>Ezh2^ΔSET/ΔSET</i> (purple) iPSC. C. Venn diagram showing overlap between H3K27me3⁺ genes in <i>Ezh2</i>^+/+ iPSC (grey), H3K27me2⁺/H3K27me3⁻ genes in <i>Ezh2^ΔSET/ΔSET</i> iPSC (orange) and H3K27me2⁺/H3K27me3⁻ in <i>Ezh2</i>^+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2⁺/H3K27me3⁺ or H3K27me2⁺/H3K27me3⁻ in <i>Ezh2^ΔSET/ΔSET</i> iPSC. For all analyses, two <i>Ezh2</i> control (+/+; grey) iPSC clones were compared to two mutant (<i>Δ</i>SET/<i>Δ</i>SET; purple) counterparts. Levels of expression are shown as ddCt (log₂ scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in <i>Ezh2</i>^+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in <i>Ezh2^ΔSET/ΔSET</i> iPSC (H3K27me3⁺ genes in green, H3K27me3⁻ genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSC depicted in panel A. Bars represent <i>P</i>-values in –Log₂ scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSC of MEF specific genes marked by H3K27me3 in <i>Ezh2</i>^+/+ iPSC. Color code is shown in panel A.</p

Mass spectrometry analysis: H3K27me3 levels below the limit of detection in <i>Ezh2^ΔSET/ΔSET</i> iPSCs.

*<p>Perkins et al 1999.</p>**<p>Cox and Mann 2010.</p><p>Mascot and PTM scores attributed in control (<i>Ezh2^+/+</i>) and mutant (<i>Ezh2^ΔSET/ΔSET</i>) iPSC clones to each combination of amino acid modifications on the H3^27–40 tri-, tetra- and penta-methylated peptide.</p

Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.

<p>A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in <i>Ezh2^+/ΔSET</i> and <i>Ezh2^ΔSET/ΔSET</i> MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in <i>Cdkn2a^−/−</i> TTF carrying either one (+/<i>Δ</i>SET) or both (<i>Δ</i>SET/<i>Δ</i>SET) <i>Ezh2</i> mutant alleles. As comparison, representative <i>Ezh2</i>-proficient (+/+) and -deficient (<i>Δ</i>SET/<i>Δ</i>SET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×10³<i>Cdkn2a^−/−</i> TTF, respectively proficient (grey bar) or deficient (purple) for <i>Ezh2</i>, assessed in three independent experiments.</p

DataSheet_1_End-of-neoadjuvant treatment circulating microRNAs and HER2-positive breast cancer patient prognosis: An exploratory analysis from NeoALTTO.pdf

BackgroundThe absence of breast cancer cells in surgical specimens, i.e., pathological complete response (pCR), is widely recognized as a favorable prognostic factor after neoadjuvant therapy. In contrast, the presence of disease at surgery characterizes a prognostically heterogeneous group of patients. Here, we challenged circulating microRNAs (miRNAs) at the end of neoadjuvant therapy as potential prognostic biomarkers in the NeoALTTO study.MethodsPatients treated within the trastuzumab arm (i.e., pre-operative weekly trastuzumab for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks; post-operative FEC for 3 cycles followed by trastuzumab up to complete 1 year of treatment) were randomized into a training (n= 54) and testing (n= 72) set. RT-PCR-based high-throughput miRNA profile was performed on plasma samples collected at the end of neoadjuvant treatment of both sets. After normalization, circulating miRNAs associated with event free survival (EFS) were identified by univariate and multivariate Cox regression model.ResultsStarting from 23 circulating miRNAs associated with EFS in the training set, we generated a 3-circulating miRNA prognostic signature consisting of miR-185-5p, miR-146a-5p, miR-22-3p, which was confirmed in the testing set. The 3-circulating miRNA signature showed a C-statistic of 0.62 (95% confidence interval [95%CI] 0.53-0.71) in the entire study cohort. By resorting to a multivariate Cox regression model we found a statistical significant interaction between the expression values of miR-194-5p and pCR status (p.interaction =0.005) with an estimate Hazard Ratio (HR) of 1.83 (95%CI 1.14- 2.95) in patients with pCR, and 0.87 (95%CI 0.69-1.10) in those without pCR. Notably, the model including this interaction along with the abovementioned 3-circulating miRNA signature provided the highest discriminatory capability with a C-statistic of 0.67 (95%CI 0.58-0.76).ConclusionsCirculating miRNAs are informative to identify patients with different prognosis among those with heterogeneous response after trastuzumab-based neoadjuvant treatment, and may be an exploitable tool to select candidates for salvage adjuvant therapy.</p