Additional file 1: of Clinical communication skills and professionalism education are required from the beginning of medical training - a point of view of family physicians

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The Contrasting Role of p16^Ink4A Patterns of Expression in Neuroendocrine and Non-Neuroendocrine Lung Tumors: A Comprehensive Analysis with Clinicopathologic and Molecular Correlations

<div><p>Lung cancer encompasses a constellation of malignancies with no validated prognostic markers. p16^Ink4A expression has been reported in different subtypes of lung cancers; however, its prognostic value is controversial. Here, we sought to investigate the clinical significance of p16^Ink4A immunoexpression according to specific staining patterns and its operational implications. A total of 502 tumors, including 277 adenocarcinomas, 84 squamous cell carcinomas, 22 large cell carcinomas, 47 typical carcinoids, 12 atypical carcinoids, 28 large cell neuroendocrine carcinomas, and 32 small cell carcinomas were reviewed and subjected to immunohistochemical analysis for p16^Ink4A and Ki67. The spectrum of p16^Ink4A expression was annotated for each case as negative, sporadic, focal, or diffuse. Expression at immunohistochemical level showed intra-tumor homogeneity, regardless tumor histotype. Enrichments in cells expressing p16^Ink4A were observed from lower- to higher-grade neuroendocrine malignancies, whereas a decrease was seen in poorly and undifferentiated non-neuroendocrine carcinomas. Tumor proliferation indices were higher in neuroendocrine tumors expressing p16^Ink4A while non-neuroendocrine malignancies immunoreactive for p16^Ink4A showed a decrease in Ki67-positive cells. Quantitative statistical analyses including each histotype and the p16^Ink4A status confirmed the independent prognostic role of p16^Ink4A expression, being a high-risk indicator in neuroendocrine tumors and a marker of good prognosis in non-neuroendocrine lung malignancies. In this study, we provide circumstantial evidence to suggest that the routinary assessment of p16^Ink4A expression using a three-tiered scoring algorithm, even in a small biopsy, may constitute a reliable, reproducible, and cost-effective substrate for a more accurate risk stratification of each individual patient.</p></div

The top 11 canonical signalling pathways influenced by constitutive PI3K signaling.

<p>Active PIK3CA (E545K)-expressing lentivirus was transduced in non-transformed lung epithelial cells (BEAS-2B). Transduced cells were selected in blasticydin, checked for expression of the exogenous PIK3CA-E545K, were analysed for their transcriptomes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030427#s2" target="_blank">Materials and methods</a>. <b>A.</b> Immunoblot for PIK3CA expression in transfected BEAS-2B cells. <b>B.</b> Heat map showing fold change patterns of DEGs induced by constitutive PI3K signalling. The heat map was generated in Matlab (Mathworks), and compares fold change patterns of DEGs in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B (p<0.01). Red: up-regulated genes; green: down-regulated genes. Fold changes of all down-regulated DEGs and all but one up-regulated DEG are #8 (central color spectrum bar). <b>C.</b> The top 11 functional categories determined by IPA, that were significantly up-regulated or down-regulated in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B are shown. The 2126 DEGs in BEAS-2B-PI3K-E545K were mapped to the IPA-defined network. The significance p-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log (p-value). <b>D.</b> Bio-functions identified by IPA in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B. <b>E.</b> Sub-Categories and Functions identified through IPA showing the genes associated to lung cancer in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B.</p

pS473 AKT immunostaining (IHC) in NSCLCs.

<p>A, left: SCC negative for pAKT phosphorylation; right: SCC positive for pS473 phosphorylation. B, left: ADC negative for pAKT phosphorylation; right: ADC positive for pS473 phosphorylation. Magnification 10× and 40×, respectively.</p

IHC and FISH analysis of AKT2 in NSCLCs.

<p>A, left: SCC negative for AKT2 expression; right: SCC positive for AKT2 expression. B, left: ADC negative for AKT2 expression; right: ADC positive for AKT2 expression. Magnification 10× and 40×, respectively. C. Dual-colour fluorescence in situ hybridization analysis of AKT2 gene copy number. FISH analysis of AKT2 (red signals) and chromosome region 19p13.1 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of AKT2 with 2 chromosome region 19p13.1 signals (gene amplification). Original magnification 100×.</p

Representation of the differential Ki67 values between p16^Ink4A-negative and p16^Ink4A-positive lung tumors.

<p>The highest differences in Ki67 indices according to p16^Ink4A expression can be observed in the poorly differentiated malignancies (<i>i</i>.<i>e</i>. small cell carcinomas and large cell carcinomas), with opposite fashions between neuroendocrine and non-neuroendocrine tumors.</p

Correlation between AKT activation (pAKT) and clinico-pathologic features of patients with OC.

a<p>Patients for which pAKT staining was available (N = 93).</p>b<p>Patients for which both Grade and pAKT staining were available (N = 87).</p>c<p>Patients for which both Figo Stage and pAKT staining were available (N = 93).</p><p><b>NS</b>: not significant.</p

AKT activation in OC.

a<p>Patients for which pAKT staining was available (N°).</p>b<p>Normal vs Tumour Tissue.</p><p><b>S-OC: S</b>erous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>E-OC: E</b>ndometrioid <b>O</b>varian <b>C</b>arcinoma.</p><p><b>CC-OC: C</b>lear <b>C</b>ell <b>O</b>varian <b>C</b>arcinoma.</p><p><b>Mu-OC: M</b>ucinous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>M-OC: M</b>ixed <b>O</b>varian <b>C</b>arcinoma.</p><p><b>NS</b>: not significant.</p

Immunostaining and gene copy number analysis of PTEN in OC.

<p>A. Left: S-OC negative for PTEN expression; right: S-OC positive for PTEN expression. B. Left: E-OC negative for PTEN expression; right: E-OC positive for PTEN expression. Magnification 40X. Magnification of the insets 10X. C. Q-RT PCR of PTEN mRNA expression in normal ovarian tissues and OC. D. Q-PCR analysis of PTEN gene copy number in normal ovarian tissues and OC. DNA from peripheral blood leukocytes (PBL) was used as reference. PTEN copy number in PBL was set arbitrarily as 2.</p

IHC and FISH analysis of PI3KCA in NSCLCs.

<p>A, left: SCC negative for PIK3CA expression; right: SCC positive for PIK3CA expression. B, left: ADC negative for PIK3CA expression; right: ADC positive for PI3KCA expression. Magnification 10× and 40×, respectively. C. Dual-color fluorescence in situ hybridization analysis of PIK3CA gene copy number. FISH analysis of PIK3CA (red signals) and chromosome region 3p14.1 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of PIK3CA with 2 chromosome region 3p14.1 signals (gene amplification). Original magnification 100×.</p