Progressive Enrichment of Stemness Features and Tumor Stromal Alterations in Multistep Hepatocarcinogenesis

<div><p>Cancer stem cells (CSCs), a subset of tumor cells, contribute to an aggressive biological behavior, which is also affected by the tumor stroma. Despite the role of CSCs and the tumor stroma in hepatocellular carcinoma (HCC), features of stemness have not yet been studied in relation to tumor stromal alterations in multistep hepatocarcinogenesis. We investigated the expression status of stemness markers and tumor stromal changes in B viral carcinogenesis, which is the main etiology of HCC in Asia. Stemness features of tumoral hepatocytes (EpCAM, K19, Oct3/4, c-KIT, c-MET, and CD133), and tumor stromal cells expressing α-smooth muscle actin (α-SMA), CD68, CD163, and IL-6 were analyzed in 36 low grade dysplastic nodules (DNs), 48 high grade DNs, 30 early HCCs (eHCCs), and 51 progressed HCCs (pHCCs) by immunohistochemistry or real-time PCR. Stemness features (EpCAM and K19 in particular) were progressively acquired during hepatocarcinogenesis in combination with enrichment of stromal cells (CAFs, TAMs, IL-6+ cells). Stemness features were seen sporadically in DNs, more consistent in eHCCs, and peaked in pHCCs. Likewise, stromal cells were discernable in DNs, showed up as consistent cell densities in eHCCs and peaked in pHCCs. The stemness features and tumor stromal alterations also peaked in less differentiated or larger HCCs. In conclusion, progression of B viral multistep hepatocarcinogenesis is characterized by an enrichment of stemness features of neoplastic hepatocytes and a parallel alteration of the tumor stroma. The modulation of neoplastic hepatocytes and stromal cells was at low levels in precancerous lesions (DNs), consistently increased in incipient cancer (eHCCs) and peaked in pHCCs. Thus, in B viral hepatocarcinogenesis, interactions between CSCs and the tumor stroma, although starting early, seem to play a major role in tumor progression.</p></div

The alteration of tumor stromal cells according to differentiation and size of HCCs.

<p>Bar charts indicate the percentage of cases expressing α-SMA (+) CAFs, CD68 (+) TAMs, CD163 (+) TAMs, and IL-6 (+) stromal cells according to differentiation (A) and size (B) of HCCs. *Statistical significance (<i>P</i> <0.05).</p

The correlation between enrichment of stemness features and tumor stromal alterations in B viral multistep hepatocarcinogenesis.

<p>Bar charts indicate the percentage of cases expressing EpCAM, K19, and CD133 protein expression according to the histoscore of CAFs (A), CD68 (+) TAMs (B), CD163 (+) TAMs (C), and IL-6 (+) stromal cells (D). *Statistical significance (<i>P</i> <0.05).</p

The expression of stemness features in B viral multistep hepatocarcinogenesis.

<p>A) Representative features of EpCAM, K19, and CD133 expression in LGDNs, HGDNs, eHCCs, and pHCCs are presented. Original magnification x200. Inset, high power magnification x400. B) Bar charts indicate the percentage of cases expressing EpCAM, K19, or CD133 protein in defined lesions of B viral multistep hepatocarcinogenesis. C) Box plots show the mRNA expression profiles of EpCAM, K19, Oct3/4, c-KIT, and c-MET in B viral multistep hepatocarcinogenesis. *Statistical significance (<i>P</i> <0.05).</p

The correlation between IL-6 (+) stromal cells and CAFs or TAMs in B viral multistep hepatocarcinogenesis.

<p>IL-6 (+) stromal cells are well correlated with α-SMA (+) CAFs (A), CD68 (+) TAMs (B), and CD163 (+) TAMs (C). *Statistical significance (<i>P</i> <0.05).</p

The alteration of tumor stromal cells in B viral multistep hepatocarcinogenesis.

<p>A) Representative features of α-SMA (+) CAFs, CD68 (+) TAMs, CD163 (+) TAMs, and IL-6 (+) stromal cells in LGDNs, HGDNs, eHCCs, and pHCCs are presented. Original magnification x200. B) Bar charts indicate the percentage of cases expressing α-SMA (+) CAFs, CD68 (+) TAMs, CD163 (+) TAMs, and IL-6 (+) stromal cells in B viral multistep hepatocarcinogenesis. *Statistical significance (<i>P</i> <0.05).</p

The expressions of stemness features according to differentiation (A, B) and size (C, D) of HCCs.

<p>Bar charts indicate the percentage of cases expressing EpCAM, K19 or CD133 protein. Box plots show the mRNA expression levels of EpCAM, K19, Oct3/4, c-KIT, and c-MET. *Statistical significance (<i>P</i> <0.05).</p

The heatmap presenting expression of EpCAM, K19, CD133, α-SMA (+) CAFs, CD68 (+) TAMs, CD163 (+) TAMs, and IL-6 (+) stromal cells in B viral multistep hepatocarcinogenesis.

<p>Histoscores of each marker are presented in the individual case of LGDNs, HGDNs, eHCCs and pHCCs.</p

Limited detection of small (≤ 10 mm) colorectal liver metastasis at preoperative CT in patients undergoing liver resection

<div><p>Objective</p><p>To retrospectively determine the sensitivity of preoperative CT in the detection of small (≤ 10 mm) colorectal liver metastasis (CRLM) nodules in patients undergoing liver resection.</p><p>Methods</p><p>The institutional review board approved the study and waived informed consent. We included 461 pathologically confirmed CRLM nodules in 211 patients (including 71 women; mean age, 66.4 years) who underwent 229 liver resections following abdominal CT. Prior to 163 resections, gadoxetic acid-enhanced liver MR imaging was also performed. Nodules were matched between pathology reports and prospective CT reports following a predefined algorithm. Per-nodule sensitivity of CT was calculated by nodule-size category. Generalized estimating equations were used to adjust for within-case correlation.</p><p>Results</p><p>Fourteen nodule sizes were missing in the pathology report. Nodules of 1–5 mm and 6–10 mm accounted for 8.1% (n = 36) and 23.5% (n = 105) of the remaining 447 nodules, and the number of nodules gradually decreased as nodule size increased beyond 10 mm. The overall sensitivity of CT was 81.2% (95% confidence interval, 77.1%, 85.2%; 365/461). The sensitivity was 8% (0%, 17%; 3/36), 55% (45%, 65%; 59/105), 91%, 95%, and 100% for nodules of 1–5 mm, 6–10 mm, 11–15 mm, 16–20 mm, and >20 mm, respectively. The nodule-size distribution was similar between resections undergoing gadoxetic acid-enhanced MR imaging and those not undergoing the MR imaging.</p><p>Conclusion</p><p>CT has limited sensitivity for nodules of ≤ 10 mm and particularly of ≤ 5 mm.</p></div

Patient flow diagram.

<p>CRLM = colorectal liver metastasis.</p