Use of Formalin-Fixed Paraffin-Embedded Samples for Gene Expression Studies in Breast Cancer Patients

<div><p>To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, <i>P</i><2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.</p></div

Comparison of reliability of GEPs obtained with Affymetrix and DASL WG platforms in FFPE samples.

<p><b>A.</b> Distribution plots of interquartile ranges for GEPs obtained from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips (left panel) or the Illumina DASL WG platform (right panel). <b>B.</b> Log₂ expression intensity levels for ESR1in 12 breast cancer FFPE samples obtained respectively using Affymetrix HG-U133 2.0 Plus chips (left panel, ESR1 probeset ID “205225_at”) or the Illumina DASL WG platform (right panel, ESR1 probe ID “3360095”), from the lowest to the highest. Black circles and grey squares dot represent respectively ER+ and ER- negative samples, as determined by IHC. <b>C.</b> Distribution plots of log₂-trasnformed fold change values obtained by Class Comparison between 6 ER+ and 6 ER- FF breast cancer samples profiled with Affymetrix HG-U133 2.0 Plus chips (left panel) or with the Illumina DASL WG platform (right panel).</p

Signal intensities, reciprocal correlations and PCA plots for GEPs from FFPE and FF samples.

<p>A. Distribution plot of signal mean intensities (grey bars) and individual detection rates (black bars) in 10 FF samples profiled with the Illumina HT-12 chips and in 10 matched FFPE samples profiled with the Illumina Ref-8 chips. Only common probes were used. B. Heat map reporting the reciprocal correlations (Pearson correlation coefficient) among GEPs obtained from 10 FF breast cancer samples using Illumina HT-12 chips (left panel) and 10 matched FFPE samples profiled with Illumina Ref-8 chips (right panel). Prior to computation of the correlations, data were separately normalized using the robust spline normalization method C. PCA plots for GEPs obtained from 10 FF breast cancer samples with the Illumina HT-12 chips (left panel) and 10 matched FFPE samples profiled with the Illumina Ref-8 chips (right panel). Red circles represent ER+ samples and bleu circles represent ER- samples. Only samples with concordant ER status by RLA and IHC were included.</p

Reciprocal correlation and PCA plots for GEPs obtained with DASL WG from 12 FFPE samples.

<p><b>A.</b> Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Illumina DASL WG platform. For each sample ER status and sample ID (to allow comparability with panel B) were also reported. <b>B.</b> PCA plots of GEPS of 12 FFPE breast cancer samples profiled using the Illumina DASL WG platform. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. Sample ID is reported.</p

sj-pdf-5-tmj-10.1177_03008916231157837 – Supplemental material for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup

Supplemental material, sj-pdf-5-tmj-10.1177_03008916231157837 for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup by Laura Vittoria, Laura Sala, Valeria Summo, Iolanda Capone, Elena Conca, Martina Toma, Joseph Ottolenghi, Francesca Testa, Umberto Cortinovis, Biagio Paolini, Antonello Cabras, Antonella Aiello and Fabio Bozzi in Tumori Journal</p

Diagnostic plots for GEPs obtained with Affymetrix arrays from 12 FFPE samples.

<p><b>A.</b> RPLC13a qPCR threshold values (determined on the cDNA fraction prior to linear amplification) as a function of present call later obtained on Affymetrix HG-U133 2.0 Plus chips for 12 FFPE breast cancer samples. <b>B.</b> Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips after MAS5 normalization. For each sample, ER status and Ct values referring to the sample-pre-assessment step with RPLC13 qPCR are also reported. <b>C.</b> PCA analysis plots of GEPS of 12 FFPE breast cancer samples profiled using the Affymetrix HG-U133 2.0 Plus chips. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. RPL13a pPCR threshold values (Ct) are also reported.</p

Biological reliability of Affmetrix FFPE data.

<p>A. Agreement between <i>ESR1</i> gene expression and IHC-derived ER status in the cohort of 44 good quality FFPE samples. B. Agreement between <i>ERBB2</i> gene expression and IHC-derived Her2 positivity in the same cohort. C. Comparison between the Genomic Grade Index and IHC-derived Ki67 positivity as independent estimators of tumor proliferation rate in the same cohort. D. Hierarchical clustering using the PAM50 gene signature. Samples belonging to the 5 molecular subtypes are indicated by dashed rectangles. Pathological definition of ER, Her2, Ki67 and grade are shown according to the color code on the right.</p

sj-pdf-1-tmj-10.1177_03008916231157837 – Supplemental material for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup

Supplemental material, sj-pdf-1-tmj-10.1177_03008916231157837 for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup by Laura Vittoria, Laura Sala, Valeria Summo, Iolanda Capone, Elena Conca, Martina Toma, Joseph Ottolenghi, Francesca Testa, Umberto Cortinovis, Biagio Paolini, Antonello Cabras, Antonella Aiello and Fabio Bozzi in Tumori Journal</p

Additional file 2: of Observational study on the prognostic value of testosterone and adiposity in postmenopausal estrogen receptor positive breast cancer patients

Figure S2. Boxplots of circulating level of testosterone (ng/mL) according to tumour histology (IDC = Invasive Ductal Carcinoma; ILC = Invasive Lobular Carcinoma), Grade (G), number of metastatic axillary lymph Nodes (N), progesterone receptor (PR) and HER-2 status of ER-positive postmenopausal breast cancer patients. Number and percentage of patients in each group are reported and p-values are given. The bar inside the box is the median value and the box upper and lower dimensions define the inter-quartile range. Shows the boxplots of circulating level of testosterone (ng/mL) according to the other tumour characteristics considered in the study (histology, tumour grade, axillary nodal status, PR and HER-2 status). On the whole, the results did not indicate an association between circulating level of testosterone and unfavorable tumour characteristics as high tumour grade, axillary involvement or HER2 overexpression. (PDF 22 kb

sj-pdf-4-tmj-10.1177_03008916231157837 – Supplemental material for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup

Supplemental material, sj-pdf-4-tmj-10.1177_03008916231157837 for Breast implant associated anaplastic large cell lymphoma: Evidence for an efficient diagnostic workup by Laura Vittoria, Laura Sala, Valeria Summo, Iolanda Capone, Elena Conca, Martina Toma, Joseph Ottolenghi, Francesca Testa, Umberto Cortinovis, Biagio Paolini, Antonello Cabras, Antonella Aiello and Fabio Bozzi in Tumori Journal</p