Cox regression analyses.

<p>fn id="t001fn001">Univariate and multivariate Cox regression analyses for FIGO stage, presence of lymph node metastasis and vaso-invasion and moderate/strong versus absent/weak tumor galectin-1 expression on disease-specific survival are shown. Bold numbers indicate a correlation with p<0.05.</p><p>Cox regression analyses.</p

Distribution of galectin expression.

<p>The fraction of samples in which tumor cells expressed galectin-1, -3 or -9 or a combination is shown in A. The fraction of samples positive for one or none of the galectin types studied is shown in the first percentages column, while fractions of samples expressing two or more galectin types are shown in the second column. Total galectin-1, -3 and -9 single, double and triple positive pixels in the tumor stroma and tumor epithelium is represented in B. Mean and standard error of the mean are displayed.</p

Survival analyses for galectin-1, -3 and -9 expression.

<p>Kaplan-Meier survival curve for strong compared with absent to weak expression of galectin-1 by tumor cells is shown in A. A survival analysis comparing patients with present versus absent tumor expression of galectin-9 is shown in B.</p

Immunofluorescent staining of galectin-1, -3 and -9.

<p>Representative triple staining image in an FFPE squamous cervical cancer sample containing tumor epithelial cells expressing galectin-1 (A, blue), galectin-3 (B, green) and galectin-9 (C, red) at a 200x magnification. Tumor epithelial fields are marked by dashed lines. Typically, galectin-1 was expressed in a high number of stromal cells and could be weakly expressed by tumor epithelial cells. Galectin-3 was expressed in stromal cells and often either in epithelial field centers (as shown here) or in small groups of epithelial cells at the invasive border. Galectin-9 could also be expressed by both stromal and epithelial cells. When galectin-1 or -9 were expressed by epithelial cells, this was typically observed at the borders of tumor fields, while galectin-3 was expressed in the center, as demonstrated in this figure. Tumor infiltrating CAS cells expressing galectin-1, -3 and -9 were also frequently observed within the tumor epithelial fields.</p

Five-year disease-free survival by mutational status per histological subtype.

<p>Five-year disease-free Kaplan-Meier survival curves for cervical squamous cell carcinoma (A), cervical adenocarcinoma (B), and cervical adenosquamous carcinoma (C) patients based on mutational status. <i>P</i>-values were calculated by the Log Rank-test.</p

Five-year disease-free survival curve by <i>CTNNB1</i> gene mutation status for cervical squamous cell carcinoma patients.

<p>The <i>P-</i>value was calculated by the Log Rank-test.</p

Examples of DNA content analysis of recurrent NMTC. Multiparameter DNA content analysis was performed on FFPE NMTC, as described.

<p><b>A</b>. Multiparameter DNA content analysis of a bimodal PTC with a DI of 1.02 and 2.05 (case No. 19), <b>B</b>. a PTC-OV with a DI of 0.97 (case No. 25) and <b>C</b>. a bi-modal FTC-OV with a DI of 0.53 and 1.04, respectively (case No. 13). <b>a</b>. Haematoxylin – eosin staining 200×. <b>b</b>. keratin vs. vimentin density plot (note the vimentin co-expression of these tumours and the clear separation between the stromal and the epithelial cell fraction. The expression of keratin and vimentin are high, relative to the controls showing background fluorescence [<b>d</b>]). Twenty-five samples, 93% (25/27), showed high vimentin co-expression in more than 50% of the cancer cells (data not shown). <b>c</b>. DNA histogram generated after gating on the epithelial cell fraction. <b>e</b>. DNA histogram generated after gating on the normal DNA diploid stromal cell fraction. This fraction was used as a DNA content reference. <b>f</b>. DNA histogram of the epithelial cell fraction after modelling by ModFit (note that the presence of a second cell cycling population in the bimodal PTC and the FTC-OV DNA histograms is significant and demonstrates endoreduplication. In addition, the FTC-OV shows a dominant DNA near-haploid population [<b>c</b>, <b>f</b>]).</p

Summary of the genomic alterations found after LAIR analysis (see Materials and Methods) in 27 recurrent NMTCs.

<p>In this heatmap, rows represent tumours and columns represent chromosomes. The first column shows the tumour number, type and DNA index (DI). The tumours have been grouped according to their subtype, with ten FTC-OV tumours in the upper group and 17 NON FTC-OV in the lower group. The combined frequency of genomic alterations for each group is indicated in a separate row. Black indicates allelic states with >2 copies and at least one B allele retained, e.g. [AABB] and [AAB]. Grey indicates allelic states of [A] and [AA]. The colours in the heatmap indicate: white, allelic state [AB] = normal heterozygous state. Dark red, allelic states [AABB], [AAABBB], etc. = amplified heterozygous states. Light red, allelic states [AAB], [AAABB], etc. = imbalanced gain. Dark blue, allelic state [A] = LOH or physical loss in the context of a diploid genome but monosomy in the context of a haploid genome. Light blue, allelic states [AA], [AAA], etc. = copy neutral LOH and amplified LOH, respectively. Notice the retention of chromosome 7 for five out of ten FTC-OV tumours (allelic states [AB]) with the remaining five showing endoreduplication with allelic states [AABB] (n = 4) or [AAB] (n = 1). The Integrative Genomics Viewer (IGV) was used to produce this image.</p

Interphase FISH analysis in relation to allelic state analysis.

<p>In FTC-OV, chromosome 6 was always observed in allelic state [A] or [AA], whereas chromosome 7 was always retained in a heterozygous state or amplified heterozygous state. To confirm these results, interphase FISH was performed for chromosomes 6 and 7. Examples are shown, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038287#pone-0038287-t001" target="_blank">Table 1</a>. <b>A</b>. FISH on normal thyroid epithelium. <b>B</b> and <b>C</b> show FTC-OV case No. 10. <b>B</b>: Allelic state analysis illustrating allelic state [A] for chromosome 6 and allelic state [AABB] for chromosome 7. <b>C</b>. Left panel: green signal, centromere 6 shows 1 copy, confirming the allelic state [A] in three of the four nuclei. Right panel: four green and four red signals representing EGFR and centromere 7, respectively and confirming the [AABB] allelic state. <b>D–E</b> show FTC-OV, case no 11. <b>D</b>. The allelic state [AAB] of chromosome 7 could not be confirmed definitively and showed a mixture of nuclei containing three green (EGFR) and three red (centromere) signals or four green and four red signals, respectively (<b>E</b>). This may be due to intra-tumour heterogeneity.</p

image_1_Indoleamine 2,3-Dioxygenase Expression Pattern in the Tumor Microenvironment Predicts Clinical Outcome in Early Stage Cervical Cancer.tif

<p>The indoleamine 2,3-dioxygenase (IDO) enzyme can act as an immunoregulator by inhibiting T cell function via the degradation of the essential amino acid tryptophan (trp) into kynurenine (kyn) and its derivates. The kyn/trp ratio in serum is a prognostic factor for cervical cancer patients; however, information about the relationship between serum levels and IDO expression in the tumor is lacking. IDO expression was studied in 71 primary and 14 paired metastatic cervical cancer samples by various immunohistochemical (IHC) techniques, including 7-color fluorescent multiparameter IHC, and the link between the concentration of IDO metabolites in serum, clinicopathological characteristics, and the presence of (proliferating) T cells (CD8, Ki67, and FoxP3) was examined. In addition, we compared the relationships between IDO1 and IFNG gene expression and clinical parameters using RNAseq data from 144 cervical tumor samples published by The Cancer Genome Atlas (TCGA). Here, we demonstrate that patchy tumor IDO expression is associated with an increased systemic kyn/trp ratio in cervical cancer (P = 0.009), whereas marginal tumor expression at the interface with the stroma is linked to improved disease-free (DFS) (P = 0.017) and disease-specific survival (P = 0.043). The latter may be related to T cell infiltration and localized IFNγ release inducing IDO expression. Indeed, TCGA analysis of 144 cervical tumor samples revealed a strong and positive correlation between IDO1 and IFNG mRNA expression levels (P < 0.001) and a significant association with improved DFS for high IDO1 and IFNG transcript levels (P = 0.031). Unexpectedly, IDO+ tumors had higher CD8⁺Ki67⁺ T cell rates (P = 0.004). Our data thus indicate that the serum kyn/trp ratio and IDO expression in primary tumor samples are not clear-cut biomarkers for prognosis and stratification of patients with early stage cervical cancer for clinical trials implementing IDO inhibitors. Rather, a marginal IDO expression pattern in the tumor dominantly predicts favorable outcome, which might be related to IFNγ release in the cervical tumor microenvironment.</p